JP2009191056A - Therapy of disorder mediated by anti-erythropoietin antibody using epo receptor agonist based on synthetic peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a prophylactic or therapeutic method of disorders mediated by anti-erythropoietin antibody, using an EPO receptor agonist based on a synthetic peptide. <P>SOLUTION: A novel peptide compound is an EPO-R agonist with a drastically increased efficacy and activity. The compound is a homodimer of a peptide monomer containing N-acetylglycine, 1-naphthylalanine, etc. and has an intramolecular disulfide bond formed between cysteine residues of the peptide monomers. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

(Field of Invention)
The present invention relates to peptide compounds that are agonists of erythropoietin receptor (EPO-R). The invention also provides treatment using such peptide compounds to treat or prevent disorders mediated by anti-erythropoietin (EPO) antibodies, such as erythroblastoma (PRCA), characterized by anti-EPO antibodies. Regarding the method. Also provided are pharmaceutical compositions comprising the peptide compounds of the invention.

(Background of the Invention)
As recombinant therapeutics are increasingly developed, the immunogenicity of these new drugs has become a concern (Schellekens H. (2003) Nephrol. Dial. Transplant 18: 1257-9). The use of recombinant human EPO to ameliorate anemia in chronic kidney disease (CDK) is a major advance over the last 20 years in managing this condition. Although this treatment is generally well tolerated, neutralization of anti-EPO antibodies can cause disorders such as PRCA (Bergrem H et al. (1993) In: Bauer C, eds. Erythropoietin : Molecular Physiology and Clinical Application. New York, NY, Marcel Dekker 1993: 266-275; Peces R et al. (1996) N. Engl J. Med. 334: 630-3; and Prabhakar SS et al. (1997) Clin. Nephrol. 47: 331-5). In 2002, an increase in this serious complication was reported (Casadeval NJ et al. (2002) N. Engl J. Med. 346: 469-75; and Gershon SK et al. (2002) N. Engl. J Med. 346: 1584-6).

PRCA refers to the condition where megakaryocytes and leukocyte precursors are usually present at normal levels, while there are few red blood cell precursors in the bone marrow. In patients with PRCA, anti-EPO antibodies neutralize not only the erythropoiesis promoter (ESA) (eg, epoetin or darbepoetin) but also the patient's endogenous EPO, so that in severe cases, erythropoietic activity is largely inhibited. In the bone marrow, erythroblasts are substantially eliminated and the reticulocyte count is <10 × 10 ⁹ / L (Rossert J et al. (2004) J. Am. Soc. Nephrol. 15: 398-406). The resulting anemia is severe, hemoglobin concentrations often fall to 5 or 6 g / dL, patients become dependent on blood transfusions, and usually require at least monthly erythrocyte transfusions.

The neutralizing anti-EPO antibody, primarily of the IgG1 or IgG4 subtype, is directed against the protein portion of the molecule. This is because deglycosylation of EPO does not abolish antibody binding (Casadevall NJ et al. (2002) N. Engl. J. Med. 346: 469-75). The development of PRCA mediated by anti-EPO antibodies is much more common in the subcutaneous (SC) use of one of the epoetin alfa formulations sold outside the United States (EPREX ^™ or ERYPO ^™ ) Yes (Bennett CL et al. (2004) N. Engl. J. Med. 351: 1385-7), the approval for subcutaneous administration of these products was temporarily withdrawn in Europe. In general, because of antigen presentation by skin Langerhans cells (dendritic cells), administration by the subcutaneous route of proteins is more likely to generate immunity. Although the use of the subcutaneous route has been reduced and subsequent formulation improvements have led to a rapid reduction in the incidence of the disease, there is still a low baseline rate and the most recently marketed ESA has been reported. (Bennett CL et al. (2004) N. Engl. J. Med. 351: 1385-7; and Howman R et al. (2007) Nephrol. Dial. Transplant 22: 1462-4). In November 2005, a “Dear Doctor” letter was issued, which was administered intravenously (IV) to all US-approved ESAs in patients undergoing hemodialysis to avoid the risk of antibody formation. ) Recommended to administer. This has significant implications for cost. This is because administration of epoetin alfa and beta by the subcutaneous route is more efficient than the intravenous route (Kaufman JS et al. (1998) N. Engl. J. Med. 339: 578-83). There are also reports of anti-EPO antibodies that form spontaneously in patients who have not received ESA treatment and cause PRCA of autoimmune disease (Casadevall N et al. (1996) N. Engl. J. Med. 334: 630-3). Furthermore, the potential risk of generating anti-EPO antibodies can also affect the development and use of ESAs, including biologically similar drugs.

  Previous treatment of PRCA mediated by anti-EPO antibodies is problematic (Verhelst D et al. (2004) Lancet 363: 1768-71). Anemia associated with PRCA mediated by anti-EPO antibodies has low reticulocyte count, low hemoglobin levels, absence of erythroblasts in the bone marrow, resistance to treatment with recombinant human EPO, and moderate to EPO Characterized by Japanese antibodies (Casadevall N et al. (1996) N. Engl. J. Med. 334: 630-3). The neutralizing effect of the antibody does not go away with increasing doses of ESA. Switching to another recombinant ESA is not appropriate due to the cross-reactivity of anti-EPO antibodies. Antibody-mediated PRCA makes it impossible for patients to routinely prevent the generation of anti-EPO antibodies to prevent further treatment with recombinant EPO, improve patient anemia, and control hemoglobin levels. Require regular blood transfusion and / or immunosuppressive therapy. In some cases, immunosuppressive therapy cures the disease (Verhelst D et al. (2004) Lancet 363: 1768-71), but these medications are associated with considerable risks and side effects. If PRCA remains untreated or does not respond to immunosuppression, patients usually continue to rely on blood transfusions, which causes iron overload. Few patients were able to receive protein-based ESA treatment again after disease recovery, which carries the risk of recurrent antibody formation (Andrade J et al. (2005) Nephrol. Dial. Transplant 20: 2548-51 ). There is also the risk of an anaphylactoid reaction after repeated injections of protein-based ESA in antibody-mediated patients with PRCA (Weber G et al. (2002) J. Am. Soc. Nephrol. 13: 2381-3 ). Therefore, a safer and more effective treatment for this syndrome is highly desirable.

  The concept that peptides can act as EPO receptor agonists and stimulate erythropoiesis was first reported in 1996, but the original peptide (EMP-l) was not developed as a therapeutic agent (Wrighton NC (1996) Science 273: 458-464). An ESA based on a novel synthetic peptide having an amino acid sequence that is completely unrelated to natural EPO and recombinant EPO has been published in U.S. Patent Application Publication No. 2005/0137329 (patented as U.S. Patent No. 7,084,245), 2007. / 0027074, 2007/0104704, US non-provisional application No. 11 / 777,500 (filed July 13, 2007), and International Publication No. WO 2006/060148, in vitro, and various animals It has been shown to stimulate erythropoiesis in species (Fan Q. et al. (2006) Exp. Hematol. 34: 1303-11).

  Treatment of disorders such as PRCA characterized by anti-EPO antibodies resulting from the use of protein-based ESAs is currently unsatisfactory due to side effects and limited immunosuppressive therapy success. The risk of PRCA has a significant impact on the management of anemia, resulting in limited use of ESA subcutaneously (SC). The latest invention provides a method of stimulating erythropoiesis in patients with anti-EPO antibodies by administering to the patient a synthetic peptide-based EPO receptor agonist. The latest invention also provides a method for preventing or treating disorders such as PRCA in patients with anti-EPO antibodies by administering a synthetic peptide-based EPO receptor agonist to the patient.

(Summary of the Invention)
The present invention provides novel peptide compounds that are EPO-R agonists with dramatically enhanced potency and activity. These peptide compounds are homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1), or the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (sequence A homodimer of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) (SEQ ID NO: 3), wherein each amino acid is a standard Indicated by a single letter abbreviation. “(AcG)” is N-acetylglycine, “(1-nal)” is 1-naphthylalanine, and “(MeG)” is N-methylglycine, also known as sarcosine. Each peptide monomer of the peptide dimer contains an intramolecular disulfide bond between the cysteine residues of the monomer.

Peptide monomers may be dimerized by covalent linkage to a branched tertiary amide linker. The tertiary amide linker is
-C ¹ O-CH ₂ -X-CH ₂ -C ² O-
Can be expressed as:
Where X is NCO— (CH ₂ ) ₂ —N ¹ H—; the linker C ¹ forms an amide bond with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; ² forms an amide bond with the ε-amino group of the C-terminal lysine residue of the second peptide monomer; N ¹ of X binds to the activated polyethylene glycol (PEG) moiety through a carbamate bond or an amide bond, where The molecular weight of PEG is about 20,000 to 40,000 daltons (the term “about” indicates that in the preparation of PEG, some molecules are larger or smaller than the stated molecular weight).

When each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) and the linker N ¹ is linked to the activated polyethylene glycol (PEG) moiety through a carbamate bond, The novel peptide compounds can be shown as follows:

When each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) and the linker N ¹ is linked to the activated polyethylene glycol (PEG) moiety through an amide bond, The novel peptide compounds can be shown as follows:

When each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), and the linker N ¹ binds to the activated polyethylene glycol (PEG) moiety through a carbamate bond The novel peptide compound of the present invention can be shown as follows:

When each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), and the linker N ¹ binds to the activated polyethylene glycol (PEG) moiety through an amide bond The novel peptide compound of the present invention can be shown as follows:

Peptide monomers may be dimerized by covalent attachment to a branched tertiary amide linker. The tertiary amide linker is
-C ¹ O-CH ₂ -X-CH ₂ -C ² O-
Can be expressed as:
Where X is NCO— (CH ₂ ) ₂ —NH—C ³ O—; the linker C ¹ forms an amide bond with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; Of C ² forms an amide bond with the ε-amino group of the C-terminal lysine residue of the second peptide monomer. The peptide dimer of the present invention has the following structure:
-N ¹ H- (CH ₂ ) ₄ -C ⁴ HN ² H-
Further comprising a spacer portion having:
Where C ⁴ of the spacer is covalently bonded to C ³ of X; N ¹ of the spacer is covalently bonded to the activated polyethylene glycol (PEG) moiety through a carbamate bond or an amide bond; N ² of the spacer is Covalently attached to the activated PEG moiety through a carbamate bond or an amide bond, where the molecular weight of PEG is about 10,000 to 50,000 daltons (the term “about” means a molecule larger than the molecular weight described in the preparation of PEG Some are small molecules). Each PEG moiety may be 10,000 daltons (1OkD), 20 kD, 30 kD, 40 kD or 50 kD, respectively.

If each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1), and both N ¹ and N ² of the spacer are covalently bonded to the activated PEG moiety through a carbamate bond, The novel peptide compounds of the invention can be shown as follows:

In a preferred embodiment, the C-terminal lysine of the two peptide monomers is L-lysine. One skilled in the art will also appreciate from the above chemical structure that the two linear PEG moieties are linked by lysine (eg, as mPEG ₂ -Lys-NHS or mPEG ₂ -ricinol-NPC) and the lysine Is also preferably L-lysine, producing the following stereochemistry.

  Alternatively, one or more lysine residues may be D-lysine, creating alternative stereochemistry, as will be readily understood by those skilled in the art.

If each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) and both the spacer N ¹ and N ² are covalently linked to the activated PEG moiety through an amide bond, The novel peptide compounds of the invention can be shown as follows:
Again, the lysine molecules in this compound are preferably all L-lysine, producing the following stereochemistry.
Alternatively, one or more lysine residues may be D-lysine, creating alternative stereochemistry, which will be readily understood by those skilled in the art.

Each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), and both spacer N ¹ and N ² are covalently linked to the activated PEG moiety through carbamate linkages Where Y is a carbamate group, the novel peptide compound of the present invention can be represented as follows:
Preferably, the lysine residues that link peptide monomers and linear PEG moieties within the molecule are all L-lysine, producing the following stereochemistry:

  Alternatively, one or more lysine residues may be D-lysine, creating alternative stereochemistry, as will be readily understood by those skilled in the art.

Each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), and both spacer N ¹ and N ² are covalently linked to the activated PEG moiety through amide bonds In this case, the novel peptide compound of the present invention can be shown as follows:
Preferably, all lysine residues that link the peptide monomer of this molecule to the linear PEG moiety are L-lysine, producing the following stereochemistry.
In other embodiments, one or more lysine residues may be D-lysine, creating alternative stereochemistry, as will be readily appreciated by those skilled in the art.

  The peptide monomer can also be dimerized by attachment to a lysine linker, whereby the first peptide monomer is linked at its C-terminus to the ε-amino group of lysine and the second peptide monomer is linked to the lysine at its C-terminus. Bonded to the α-amino group.

The peptide dimer of the present invention has the following structure:
-N ¹ H- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -N ² H-
Further comprising a spacer portion having
At one end, the spacer N ¹ is attached to the carbonyl carbon of the lysine linker through an amide bond. At the opposite end, the spacer N ² is attached to the activated polyethylene glycol (PEG) moiety through a carbamate or amide bond, where the molecular weight of the PEG is about 20,000-40,000 daltons (the term “about” "Indicates that in the preparation of PEG, some molecules are larger or smaller than the stated molecular weight).

When the spacer is attached to the activated polyethylene glycol (PEG) moiety through a carbamate linkage, the novel peptide compound of the present invention (SEQ ID NO: 3) can be represented as follows:

When the spacer is attached to the activated polyethylene glycol (PEG) moiety through an amide bond, the novel peptide compound of the present invention (SEQ ID NO: 3) can be represented as follows:

The present invention further provides methods for treating various medical conditions using such peptide compounds. Included is a method of treating or overcoming a disorder in a patient characterized by neutralizing anti-EPO antibodies by administering to the patient a therapeutically effective amount of one of the above compounds. Also included are methods of preventing or reducing the onset of disorders in patients characterized by neutralizing anti-EPO antibodies by administering to the patient a therapeutically effective amount of one of the above compounds. Also included is a method of treating or preventing a disorder characterized by a neutralizing anti-EPO antibody in a patient being treated with protein-based ESA, the method comprising administering to the patient a therapeutically effective amount of one of the above compounds. Administering. In some embodiments, the therapeutically effective amount is a dose of 0.05 to 0.3 mg of compound per kg patient body weight. Also included is a method of treating or preventing a disorder characterized by neutralizing anti-EPO antibodies in a patient who has not been treated with protein-based ESA, the method comprising administering to the patient a therapeutically effective amount of one of the above compounds. Administering. Also included are methods for ameliorating anemia in patients with disorders characterized by anti-EPO antibodies. Also included is a method of increasing or restoring hemoglobin without support by transfusion. In certain embodiments, the hemoglobin range increases or recovers to the target range of 10-13 g / dL, preferably 11-12 g / dL, without support by transfusion. Also included are methods of restoring or increasing the reticulocyte count. In certain embodiments, reticulocyte count is restored or increased to ^{100 × 10 9 / L~250 × 10} 9 / L. In certain embodiments, the disorder is PRCA.

  Further, in certain embodiments, the disorder is PRCA and the therapeutically effective amount is a dose of 0.05 to 0.3 mg of compound per kg of patient body weight. In other embodiments, the disorder to be prevented or reduced in incidence is PRCA, and the therapeutically effective amount is a dose of 0.05 to 0.3 mg of compound per kg of patient body weight. In another embodiment, the disorder is PRCA and the therapeutically effective amount is a dose of 0.075-0.2 mg compound per kg patient body weight. A therapeutically effective amount can be administered once every 2-4 weeks. In certain embodiments, the therapeutically effective amount can be administered once every four weeks (Q4W). In some embodiments, a therapeutically effective amount can be initially administered once every four weeks and additional therapeutically effective amounts can be administered every other week.

  The present invention further provides a pharmaceutical composition comprising such a peptide compound. In certain embodiments, the molecular weight of PEG is about 20,000 daltons. In other embodiments, the pharmaceutical composition comprises any one of the above compounds and a pharmaceutically acceptable carrier.

(Detailed description of the invention)
Definition :
Amino acid residues in the peptides are abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic acid Is Asp or D; glutamic acid is Glu or E; cysteine is Cys or C; tryptophan is Trp or W; arginine is Arg or R; glycine is Gly or G. Unconventional amino acids in peptides are abbreviated as follows: 1-naphthylalanine is 1-nal or Np, N-methylglycine (also known as sarcosine) is MeG or Sc; acetylated glycine (N-acetylglycine ) AcG.

  As used herein, the term “polypeptide” or “protein” refers to a polymer composed of amino acid monomers that are alpha amino acids linked together through amide bonds. Thus, the length of the polypeptide is at least 2 amino acid residues long and is usually longer. In general, the term “peptide” refers to a polypeptide that is only 2-3 amino acid residues in length. The length of the novel EPO-R agonist peptides of the present invention is preferably only about 50 amino acid residues long. More preferably, it is about 17-40 amino acid residues long. In contrast to a peptide, a polypeptide may contain any number of amino acid residues. Thus, the term “polypeptide” also includes longer amino acid sequences with peptides.

  As used herein, the phrase “pharmaceutically acceptable” means “generally considered to be safe” (eg, physiologically acceptable, gastric upset, dizziness when administered to a human). Molecular entities and compositions that do not typically cause allergic reactions or similar adverse reactions such as Preferably, as used herein, the term “pharmaceutically acceptable” has been approved by a federal or state regulatory agency for use in animals, particularly humans, or has the US Pharmacopoeia or other It means being listed in a generally approved pharmacopoeia. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water or aqueous solutions, saline, aqueous dextrose solutions and aqueous glycerol solutions are preferably used as carriers for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

  As used herein, the term “agonist” refers to an organism that binds to a complementary biologically active receptor and causes a biological response at the receptor or enhances an existing biological activity of the receptor. Biologically active ligand that activates a biologically active receptor.

Novel peptides that are EPO-R agonists The present invention provides novel peptide compounds that are EPO-R agonists with dramatically enhanced potency and activity. These peptide compounds are homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1), or the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (sequence Peptide monomer homodimer having the number 2), where each amino acid is indicated by a standard single letter abbreviation. “(AcG)” is N-acetylglycine, “(1-nal)” is 1-naphthylalanine, and “(MeG)” is N-methylglycine, also known as sarcosine. Each peptide monomer of the peptide dimer contains an intramolecular disulfide bond between the cysteine residues of the monomer. Such monomers can be shown schematically as follows:

These monomeric peptides are dimerized to provide peptide dimers with enhanced EPO-R agonist activity. The linker (L _k ) moiety is a branched tertiary amide that bridges the C-termini of two peptide monomers by simultaneous attachment to the C-terminal lysine residue of each monomer. The tertiary amide linker is
-C ¹ O-CH ₂ -X-CH ₂ -C ² O-
Can be expressed as:
Where X is NCO— (CH ₂ ) ₂ —N ¹ H—; the linker C ¹ forms an amide bond with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; ² forms an amide bond with the ε-amino group of the C-terminal lysine residue of the second peptide monomer; N ¹ of X binds to the activated polyethylene glycol (PEG) moiety through a carbamate bond or an amide bond, where The molecular weight of PEG is about 20,000 to 40,000 daltons (the term “about” indicates that in the preparation of PEG, some molecules are larger or smaller than the stated molecular weight).

The tertiary amide linker is
-C ¹ O-CH ₂ -X-CH ₂ -C ² O-
Can be expressed as:
Where X is NCO— (CH ₂ ) ₂ —NH—C ³ O—; the linker C ¹ forms an amide bond with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; Of C ² forms an amide bond with the ε-amino group of the C-terminal lysine residue of the second peptide monomer. The peptide dimer of the present invention has the following structure:
-N ¹ H- (CH ₂ ) ₄ -C ⁴ HN ² H-
Further comprising a spacer portion having:
Here, spacer C ⁴ is covalently bonded to C ³ of X; spacer N ¹ is covalently bonded to the activated PEG moiety through a carbamate bond or amide bond; spacer N ² is a carbamate bond or amide Covalently attached to the activated PEG moiety through conjugation, where the molecular weight of PEG is about 10,000-60,000 daltons (the term “about” means that in the preparation of PEG, some molecules are larger or smaller than the molecular weights described. Indicates that there are molecules).

  Thus, the novel peptides of the present invention can also contain a PEG moiety, which is covalently linked to the tertiary amide linker of the peptide dimer through a carbamate bond or an amide bond. PEG is a pharmaceutically acceptable water-soluble polymer. A PEG for use in the present invention may be a linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20K) to about 60K (the term “about” means in the preparation of PEG, Indicating that some molecules are larger than the molecular weight listed and some are smaller). Most preferably, the molecular weight of PEG is about 30K-40K. One skilled in the art will be able to select the desired polymer size based on considerations of: desired dose; time to circulate; resistance to proteolysis; effects on biological activity, if any Ease of handling; degree or lack of antigenicity; and other known effects of PEG on therapeutic peptides.

  The peptides, peptide dimers, and other peptide-based molecules of the present invention can be water soluble using any of a variety of chemical reactions that attach a water soluble polymer to the receptor binding portion of the molecule (eg, peptide + spacer). It can be conjugated to a polymer (eg PEG). Typical embodiments use a single point of attachment for the covalent attachment of the water soluble polymer and the receptor binding moiety, but in other embodiments, multiple points of attachment can be used. This includes further variations where different types of water soluble polymers bind to the receptor binding moiety at different points of attachment. This may include a spacer and / or a covalent point of attachment to one or both peptide chains. In some embodiments, the dimer or higher order multimer will comprise different types of peptide chains (ie, heterodimers or other heteromultimers). By way of example (but not limited to), the dimer may comprise a first peptide chain having a PEG attachment point, and the second peptide chain lacks a PEG attachment point or the first Different bond chemistries from the peptide chain may be utilized, and in some variations, the spacer may contain or lack a PEG attachment point, and if the spacer is PEGylated, A chemical reaction of a bond different from that of the first and / or second peptide chain may be used. Another embodiment uses a PEG that binds to the spacer portion of the receptor binding moiety and a different water soluble polymer (eg, a carbohydrate) that binds to one side chain of an amino acid of the peptide portion of the molecule.

  A wide variety of polyethylene glycol (PEG) can be used for PEGylation of the receptor binding moiety (peptide + spacer). Virtually any suitable reactive PEG reagent can be used. In a preferred embodiment, the reactive PEG reagent will bind to the receptor binding moiety to form a carbamate bond or an amide bond. Appropriate reactive PEG types are listed in the drug delivery system catalog (2003) of NOF Corporation (4-20-3 Ebisu Garden Place Tower, Shibuya-ku, Tokyo 150-6019) and Nektar Therapeutics (35806 Huntsville, Alabama) Including but not limited to those listed in the Molecular Engineering Catalog (2003) of Drive 490). By way of example (but not limited to), the following PEG reagents are often preferred in various embodiments: mPEG2-NHS; mPEG2-ALD; multi-Arm PEG; mPEG (MAL) 2 MPEG2 (MAL); mPEG-NH2; mPEG-SPA; mPEG-SBA; mPEG-thioester; mPEG-double ester; mPEG-BTC; mPEG-ButyrALD; mPEG-ACET; heterofunctional PEG (NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-VS, NHS-PEG-MAL); PEG acrylate (ACRL-PEG-NHS); PEG-phospholipid (eg mPEG-DSPE); selected by those skilled in the art SUNBRITE series branched PEG including GL series of glycerin-based PEG activated by chemical reaction; any of SUNBRITE activated PEG (carboxyl-PEG, p-NP-PEG, Tresyl-PEG, aldehyde PEG, acetal- PEG, amino-PEG, thiol-PEG, maleimide-PEG, hydroxyl-PEG-amine, amino-PEG-COOH, hydroxyl-PEG-aldehyde, carboxylic anhydride Type-PEG, including functionalized PEG- phospholipid, but not limited to); and the particular application and usage for other similar that one skilled in the art selects and / or suitable reactive PEG.

  The novel peptides of the invention may also contain two PEG moieties that are covalently attached to the spacer moiety through a carbamate or amide bond, where the spacer moiety is covalently attached to the tertiary amide linker of the peptide dimer. . Each of the two PEG moieties used in such embodiments of the invention may be linear and may be joined together at a single point of attachment. The molecular weight of each PEG moiety is preferably about 10 kilodaltons (10K) to 60K (the term “about” means that some molecules are larger or smaller than the molecular weights described in the preparation of PEG. Show). A linear PEG moiety is particularly preferred. More preferably, the molecular weight of each of the two PEG moieties is from about 20K to 40K, more preferably between about 20K and about 40K. More preferably, the molecular weight of each of the two PEG moieties is about 20K. One skilled in the art will be able to select the desired polymer size based on considerations of: desired dose; time to circulate; resistance to proteolysis; effects on biological activity, if any Ease of handling; degree or lack of antigenicity; and other known effects of PEG on therapeutic peptides.

The present invention also includes a peptide agonist that is a homodimer of a peptide monomer having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) (SEQ ID NO: 3), wherein each amino acid is a standard single letter Indicated by abbreviation. “(AcG)” is N-acetylglycine, “(1-nal)” is 1-naphthylalanine, and “(MeG)” is N-methylglycine, also known as sarcosine. Each peptide monomer of the peptide dimer contains an intramolecular disulfide bond between the cysteine residues of the monomer. Such monomers can be shown schematically as follows:

These monomeric peptides are dimerized to provide peptide dimers with enhanced EPO-R agonist activity. The linker (L _k ) moiety is a lysine residue that bridges the C-terminus of two peptide monomers by simultaneous attachment to the C-terminal amino acid of each monomer. One peptide monomer binds to the ε-amino group of lysine at its C-terminus, and the second peptide monomer binds to the α-amino group of lysine at its C-terminus. For example, dimers can be shown structurally as in formula I and summarized as in formula II:
In Formula I and Formula II, N ² represents the nitrogen atom of the ε-amino group of lysine, and N ¹ represents the nitrogen atom of the α-amino group of lysine.

The peptide dimer of the present invention has the following structure:
-N ¹ H- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -N ² H-
Further comprising a spacer portion having
At one end, the spacer N ¹ is attached to the carbonyl carbon of the lysine linker through an amide bond. At the opposite end, the spacer N ² is attached to the activated polyethylene glycol (PEG) moiety through a carbamate or amide bond, where the molecular weight of PEG is about 10,000-60,000 daltons (the term “about” "Indicates that in the preparation of PEG, some molecules are larger and smaller than the stated molecular weight). More preferably, the molecular weight of PEG is between about 20,000 and 40,000 daltons.

  Thus, the novel peptides of the present invention also contain a PEG moiety, which is covalently linked to the peptide dimer. PEG is a pharmaceutically acceptable water-soluble polymer. A PEG for use in the present invention may be a linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20K) to about 60K (the term “about” means in the preparation of PEG, Indicating that some molecules are larger than the molecular weight listed and some are smaller). Most preferably, the molecular weight of PEG is from about 20K to about 40K, and more preferably the molecular weight is from about 30K to about 40K. One skilled in the art will be able to select the desired polymer size based on considerations of: desired dose; time to circulate; resistance to proteolysis; effects on biological activity, if any Ease of handling; degree or lack of antigenicity; and other known effects of PEG on therapeutic peptides.

When each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) and the linker N ¹ is linked to the activated polyethylene glycol (PEG) moiety through a carbamate bond, The novel peptide compounds can be shown as follows:

When each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) and the linker N ¹ is linked to the activated polyethylene glycol (PEG) moiety through an amide bond, The novel peptide compounds can be shown as follows:

When each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), and the linker N ¹ binds to the activated polyethylene glycol (PEG) moiety through a carbamate bond The novel peptide compound of the present invention can be shown as follows:

When each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), and the linker N ¹ binds to the activated polyethylene glycol (PEG) moiety through an amide bond The novel peptide compound of the present invention can be shown as follows:

Preferred peptide dimers of the present invention include, but are not limited to:

If each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1), and both N ¹ and N ² of the spacer are covalently bonded to the activated PEG moiety through a carbamate bond, The novel peptide compounds of the invention can be shown as follows:

If each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) and both the spacer N ¹ and N ² are covalently linked to the activated PEG moiety through an amide bond, The novel peptide compounds of the invention can be shown as follows:

Each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), and both spacer N ¹ and N ² are covalently linked to the activated PEG moiety through carbamate linkages In this case, the novel peptide compound of the present invention can be shown as follows:

Each monomer of the homodimer has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), and both spacer N ¹ and N ² are covalently linked to the activated PEG moiety through amide bonds In this case, the novel peptide compound of the present invention can be shown as follows:

Preferred peptide dimers of the present invention include, but are not limited to:

When the spacer is attached to the activated polyethylene glycol (PEG) moiety through a carbamate linkage, the novel peptide compound of the present invention (SEQ ID NO: 3) can be represented as follows:

When the spacer is attached to the activated polyethylene glycol (PEG) moiety through an amide bond, the novel peptide compound of the present invention (SEQ ID NO: 3) can be represented as follows:

This dimeric structure can be written as [ _Ac- peptide, disulfide] ₂ Lys-spacer-PEG _20-40K and shows the following: the N-terminus that binds to both the α and ε amino groups of lysine Acetylated peptides, each peptide containing an intramolecular disulfide loop and a spacer molecule forming a covalent bond between the C-terminus of lysine and the PEG moiety, wherein the molecular weight of PEG is about 20,000 to about What is 40,000 daltons.

Preferred peptide dimers of the present invention include, but are not limited to:

  20 conventional amino acid stereoisomers (eg D-amino acids), non-natural amino acids such as a, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids It can be a suitable component of the compounds of the invention. Examples of non-conventional amino acids include, but are not limited to: β-alanine; 3-pyridylalanine; 4-hydroxyproline; O-phosphoserine; N-methylglycine; N-acetylserine; -Formylmethionine; 3-methylhistidine; 5-hydroxylysine; norleucine; and other similar amino acids and imino acids. Other mutations are possible, including, for example: amino terminal mutations; carboxy terminal mutations; substitution of one or more naturally-occurring genetically encoded amino acids with non-conventional amino acids; one Side chain mutations of the above amino acid residues; peptide phosphorylation and the like.

  The peptide sequences of the invention can be present alone or in combination with an N-terminal and / or C-terminal extension of the peptide chain. Such an extension may be a naturally encoded peptide sequence optionally with or without substantial non-natural sequence. The extensions may include any additions, deletions, point mutations, or other sequence variations or combinations as desired by those skilled in the art. For example (but not limited to), the natural sequence may be full length or partial length, and includes a site for binding a carbohydrate, PEG, or other polymer through a side chain bond. Amino acid substitutions to provide may be included. In some variations, amino acid substitutions result in humanized sequences that are compatible with the human immune system. All types of fusion proteins are provided, including immunoglobulin sequences adjacent to or in close proximity to the EPO-R activation sequences of the present invention with or without non-immunoglobulin spacer sequences. One type of embodiment is an immunoglobulin chain having an EPO-R activation sequence in place of the variable (V) region of the heavy and / or light chain.

Preparation of peptide compounds of the present invention (peptide synthesis)
The peptides of the present invention can be prepared by classical methods known in the art. These standard methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis, and DNA recombination techniques (eg, Merrifield J. Am. Chem. Soc. 1963 85: 2149 reference).

  In one embodiment, peptide monomers of the peptide dimer are synthesized individually and dimerized after synthesis.

In another embodiment, the dimeric peptide monomer has two functional groups capable of functioning as the initiation site for peptide synthesis and another molecular moiety (eg, may be present on the solid support surface). Linked through the C-terminus by a branched tertiary amide linker L _K moiety having a third functional group (eg, a carboxyl group or an amino group) that allows coupling. In this case, the variation of solid phase synthesis techniques, two peptide monomers may be synthesized directly onto two reactive nitrogen groups of the linker L _K moiety. Such synthesis may be sequential or simultaneous.

In another embodiment, the variation of solid phase synthesis techniques, two peptide monomers may be synthesized directly onto two reactive nitrogen groups of the linker L _K moiety. Such synthesis may be sequential or simultaneous. In this embodiment, two amino groups capable of functioning as initiation sites for peptide synthesis and binding to another molecular moiety (eg, which may be present on the solid support surface). A lysine linker (L _K ) moiety having a trifunctional group (eg, a lysine carboxyl group; or an amino group of lysine amide, a lysine residue in which the carboxyl group is converted to the amide moiety —CONH ₂ ) is used.

When the sequential synthesis of the dimer peptide chain on the linker is to be performed, the two amine functional groups on the linker molecule are protected with two different orthogonally removed amine protecting groups. The protected linker is attached to the solid support through the third functional group of the linker. The primary amine protecting group is removed and the dimeric first peptide is synthesized on the first deprotected amine moiety. The secondary amine protecting group is then removed and a dimeric second peptide is synthesized on the second deprotected amine moiety. For example, the first amino portion of the linker may be protected with Alloc and the second amino portion may be protected with Fmoc. In this case, the Fmoc group (not the Alloc group) can be removed by treatment with a weak base (eg 20% piperidine in dimethylformamide (DMF)), and the first peptide chain is synthesized. The Alloc group can then be removed using an appropriate reagent (eg, Pd (PPh ₃ ) / 4-methylmorpholine and chloroform), and a second peptide chain is synthesized. Use this technique when different cysteine thiol protecting groups should be used to control disulfide bond formation (described below) even if the final amino acid sequence of the dimer peptide chain is identical Note that there is a need to do.

  If simultaneous synthesis of the dimer peptide chain onto the linker is to be performed, the two amine functional groups of the linker molecule are protected with the same removable amine protecting group. The protected linker is attached to the solid support through the third functional group of the linker. In this case, the two protected functional groups of the linker molecule are simultaneously deprotected and two peptide chains are synthesized simultaneously on the deprotected amine. Note that using this technique, the sequence of the dimer peptide chain is identical and the thiol protecting groups of the cysteine residues are all identical.

  A preferred method for peptide synthesis is solid phase synthesis. Solid phase peptide synthesis procedures are well known in the art (eg, Stewart Solid Phase Peptide Syntheses (Freeman and Co .: San Francisco) 1969; Novabiochem, San Diego, USA 2002/2003 General Catalog; Goodman Synthesis of Peptides and Peptidomimetics (Houben -Weyl, Stuttgart (see 2002). In solid phase synthesis, synthesis typically begins at the C-terminal end of the peptide using an α-amino protected resin. Suitable starting materials can be prepared, for example, by attaching the required α-amino acid to a chloromethylated resin, a hydroxymethyl resin, a polystyrene resin, or a benzhydrylamine resin. One such chloromethylated resin is commercially available under the trade name BIO-BEADS SX-1 (manufactured by Bio Rad Laboratories, Richmond, Calif.). The preparation of hydroxymethyl resins has been described (Bodonszky et al. (1966) Chem. Ind. London 38: 1597). Benzhydrylamine (BHA) resin has been described (Pietta and Marshall (1970) Chem. Commun. 650) and the hydrochloride salt form is commercially available from Beckman Instruments (Palo Alto, Calif.). For example, according to the method described in Gisin (1973) Helv. Chim. Acta 56: 1467, α-amino protected amino acids can be coupled to chloromethylated resins with the aid of a cesium bicarbonate catalyst.

  After the first coupling, the α-amino protecting group is removed using, for example, a solution of trifluoroacetic acid (TFA) or hydrochloric acid (HCl) in an organic solvent at room temperature. The α-amino protected amino acid is then sequentially bound to the increasing support-bound peptide chain. Alpha amino protecting groups are known to be useful in the field of stepwise synthesis of peptides and include: acyl-type protecting groups (eg formyl, trifluoroacetyl, acetyl); aromatic urethane type Protecting groups (eg benzyloxycarboyl (Cbz) and substituted Cbz); aliphatic urethane protecting groups (eg t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl); alkyl-type protecting groups (eg benzyl , Triphenylmethyl); fluorenylmethyloxycarbonyl (Fmoc); allyloxycarbonyl (Alloc); and 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) .

  Side chain protecting groups (typically ethers, esters, trityl, PMC, etc.) remain intact during attachment and are not separated during deprotection or coupling of the amino terminal protecting group. Side chain protecting groups must be removable when the synthesis of the final peptide is complete and do not alter the target peptide under the reaction conditions. Tyr side-chain protecting groups include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. Asp side chain protecting groups include benzyl, 2,6-dichlorobenzyl, methyl, ethyl and cyclohexyl. Thr and Ser side chain protecting groups include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl and Cbz. Arg side chain protecting groups are nitro, tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), 4 Contains -methoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr) or Boc. Lys side chain protecting groups include Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tos or Boc.

After removal of the α-amino protecting group, the remaining protected amino acids are coupled stepwise in the desired order. Each protected amino acid is typically 2- (1H-benzotriazole in solution (eg, methylene chloride (CH ₂ Cl ₂ ), N-methylpyrrolidone, dimethylformamide (DMF), or mixtures thereof). The reaction is carried out in about a 3-fold excess using a suitable carboxyl group activator such as 1-yl) -1,1,3,3 tetramethyluronium hexafluorophosphate (HBTU) or dicyclohexylcarbodiimide (DCC).

  After completion of the desired amino acid sequence, the desired peptide is separated from the resin support by treatment with a reagent such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF), and the peptide is not only cleaved from the resin. All remaining side chain protecting groups are also cleaved. When using chloromethylated resins, treatment with hydrogen fluoride results in the formation of free peptide acids. When using a benzhydrylamine resin, treatment with hydrogen fluoride results in the free peptide amide directly. Alternatively, when a chloromethylated resin is employed, the peptide with side chains protected can be decoupled by treating the peptide resin with ammonia, giving the desired side chain protected amide. . Alternatively, treatment with an alkylamine provides a side chain protected alkylamide or dialkylamide. The side chain protection is then removed in the usual manner by treatment with hydrogen fluoride to give the free amide, alkyl amide, or dialkyl amide.

  In the preparation of the esters of the present invention, the resin used to prepare the peptide acid is used and the side chain protected peptide is cleaved using a base and a suitable alcohol (eg, methanol). The side chain protecting group is then removed in the usual manner by treatment with hydrogen fluoride to give the desired ester.

  These procedures are peptides of any of the compounds of the present invention, wherein amino acids other than the 20 natural genetically encoded amino acids are substituted at one, two or more positions. Can also be used to synthesize. Synthetic amino acids that can be substituted for the peptides of the present invention include, but are not limited to: N-methyl; L-hydroxypropyl; L-3,4-dihydroxyphenylalanyl; δ amino acid (L-δ-hydroxylysyl) And D-δ-methylalanyl and the like); L-α-methylalanyl; β amino acids; and isoquinolyl. D-amino acids and non-natural synthetic amino acids can also be incorporated into the peptides of the present invention.

(Modification of peptide)
In addition, the amino terminus and / or carboxy terminus of the peptide compound of the present invention can be modified to produce other compounds of the present invention. For example, the amino terminus can be acetylated using acetic acid or a halogenated derivative thereof (such as α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid).

  The natural side chains of the 20 genetically encoded amino acids (or stereoisomeric D-amino acids) can be replaced with: other side chains such as alkyl, lower alkyl, 4, 5, 6 Or a group such as a 7-membered cyclic alkyl, amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy and a lower ester derivative thereof; and a 4, 5, 6 or 7-membered heterocyclic ring. In particular, a proline analog in which the ring size of the proline residue is modified from a 5-membered ring to a 4, 6- or 7-membered ring can be employed. The cyclic group may be saturated or unsaturated, and when unsaturated, may be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen and / or sulfur heteroatoms. Examples of such groups include: furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (eg morpholino), oxazolyl, piperazinyl (eg 1-piperazinyl), piperidyl (eg 1-piperidyl, piperidino) , Pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (eg 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (eg thiomorpholino), and triazolyl. These heterocyclic groups may or may not be substituted. If the group is substituted, the substituent may be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

  In addition, peptides can be easily modified by phosphorylation and other methods (eg, as described in Hruby et al. (1990) Biochem J. 268: 249-262).

  The peptide compounds of the present invention also serve as structural models for non-peptidic compounds with similar biological activity. In order to construct compounds that have the same or similar desired biological activity as the lead peptide compound, but have a more favorable activity than the lead compound in terms of solubility, stability and sensitivity to hydrolysis and proteolysis. Those skilled in the art recognize that the following techniques are available (see Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24: 243-252). These techniques involve replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines and N-methylamino acids.

(Disulfide bond formation)
The compounds of the present invention contain two intramolecular disulfide bonds. Such a disulfide bond can be formed by oxidation of a cysteine residue of each peptide monomer.

In one embodiment, control of cysteine bond formation is accomplished by selecting an effective type and concentration of oxidant for optimal formation of the desired isomer. For example, oxidation of peptide dimers to form two intramolecular disulfide bonds (one for each peptide chain) is preferentially achieved when the oxidizing agent is DMSO or iodine (I ₂ ) (intramolecular Rather than disulfide bond formation).

  In other embodiments, cysteine bond formation is controlled by selective use of thiol protecting groups during peptide synthesis. For example, if a dimer having two intramolecular disulfide bonds is desired, the first monomeric peptide chain is linked to a first thiol protecting group (eg, trityl (Trt), allyloxycarbonyl (Alloc), and 1- (4,4 -Dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) and the like) and the second monomeric peptide is It is synthesized using two cysteine residues in the core sequence protected with a second thiol protecting group (eg, acetamidomethyl (Acm), t-butyl (tBu), etc.) different from the 1 thiol protecting group. Thereafter, the first thiol protecting group is removed to cause cyclization of the first monomer disulfide, and then the second thiol protecting group is removed to result in cyclization of the second monomer disulfide. Let

Other embodiments of the present invention provide analogs of these disulfide derivatives, in which one of the sulfur atoms is replaced with a CH ₂ group or other sulfur isomer. These analogs can be prepared from the compounds of the present invention, wherein each peptide monomer contains at least one C or homocysteine residue and is a molecule using methods known in the art. The 2C residue is replaced with α-amino-γ-butyric acid by intra- or intermolecular substitution (eg, Barker et al. (1992) J. Med. Chem. 35: 2040-2048; and Or et al. ( 1991) J. Org. Chem. 56: 3146-3149). One skilled in the art will readily appreciate that this substitution can also occur using other homologs of α-amino-γ-butyric acid and homocysteine.

  In addition to the cyclization methods described above, other non-disulfide peptide cyclization methods can also be used. Such other cyclization methods include, for example, amide-cyclization methods and those involving the formation of thio-ether bonds. Thus, the compounds of the present invention can exist in cyclized forms having either intramolecular amide bonds or intramolecular thio-ether bonds. For example, the peptide may be synthesized such that one cysteine in the core sequence is replaced with lysine and the second cysteine is replaced with glutamic acid. Thereafter, a cyclic monomer may be formed through an amide bond between the side chains of these two residues. Alternatively, the peptide may be synthesized such that one cysteine in the core sequence is replaced with lysine (or serine). A cyclic monomer may be formed through a thio-ether bond between the side chain of the lysine (or serine) residue of the core sequence and the side chain of the second cysteine residue. Thus, in order to cyclize the compound of the present invention, in addition to the disulfide cyclization method, either the amide cyclization method or the thio-ether cyclization method can be easily used. Alternatively, the amino terminus of the peptide can be capped with α-substituted acetic acid, where the α substituent is a leaving such as α-haloacetic acid (eg, α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid). It is a group.

(Addition of branched tertiary amide linker)
Peptide monomers can be dimerized by a branched tertiary amide linker moiety. In one embodiment, the linker is incorporated into the peptide during peptide synthesis. For example, the linker L _K moiety, and two functional groups capable of serving as initiation sites for peptide synthesis, one or more of one or more other functional groups (e.g. carboxyl group that enables binding to other molecular moiety Or an amino group), the linker can be attached to the solid support. Thereafter, the variation of the solid phase synthesis technique, the two peptide monomers may by synthesized directly onto the two reactive nitrogen groups of the linker L _K moiety.

  In an alternative embodiment, the linker can be attached to the two peptide monomers of the peptide dimer after peptide synthesis. Such binding can be achieved by methods well established in the art. In one embodiment, the linker contains two functional groups suitable for attachment to the target functional group of the synthesized peptide monomer. For example, a linker containing two carboxyl groups can be reacted with the amine group of the target lysine side chain of each of the two peptide monomers in the presence of a pre-activated or appropriate coupling reagent. .

For example, peptide monomers can be converted to tertiary amide linkers:
A ^* -C ¹ O-CH ₂ -X-CH ₂ -C ² OB ^*
Where can be chemically bonded to:
X is NCO— (CH ₂ ) ₂ —NH—Y, Y is a suitable protecting group (such as t-butyloxycarbonyl (Boc) protecting group); A ^* is a suitable functional group (N -Oxysuccinimide, etc.), used to attach the linker C ¹ to the ε-amino group of the C-terminal lysine residue of the first peptide monomer; B ^* is an appropriate functional group (eg N-oxysuccinimide) , Used to attach the linker C ² to the ε-amino group of the C-terminal lysine residue of the second peptide monomer.

In addition, for example, the peptide monomer can be a tertiary amide linker:
A ^* -C ¹ O-CH ₂ -X-CH ₂ -C ² OB ^*
Where can be chemically bonded to:
X is NCO— (CH ₂ ) ₂ —NH—C ³ O—, A ^* is a suitable functional group (such as N-oxysuccinimide), and the linker C ¹ is the C-terminal lysine of the first peptide monomer Used to attach to the ε-amino group of the residue; B ^* is a suitable functional group (such as N-oxysuccinimide) and the linker C ² is attached to the C-terminal lysine residue of the second peptide monomer. A tertiary amide linker is used as a spacer moiety:
Y-NH- (CH ₂ ) ₄ -C ⁴ H-NH-Y
Chemically bonded to, where
C ³ of X is covalently bonded to C ⁴ of the spacer, and Y is a suitable protecting group (such as a t-butyloxycarbonyl (Boc) protecting group).

(Addition of lysine linker)
Peptide monomers may dimerized by a lysine linker L _K moiety. In one embodiment, a lysine linker is incorporated into the peptide during peptide synthesis. For example, content lysine linker L _K moiety, and two functional groups capable of serving as initiation sites for peptide synthesis and a third functional group that enables binding to another molecular moiety (e.g., a carboxyl group or an amino group) If so, the linker can be attached to a solid support. Thereafter, the variation of the solid phase synthesis technique, the two peptide monomers may by synthesized directly onto the two reactive nitrogen groups of the lysine linker L _K moiety.

In an alternative embodiment when the peptide dimer is dimerized by a lysine linker L _K moiety, said linker may after peptide synthesis, can be attached to the two peptide monomers of a peptide dimer. Such binding can be achieved by methods well established in the art. In one embodiment, the linker contains at least two functional groups suitable for attachment to the target functional group of the synthesized peptide monomer. For example, the two free amine groups of lysine can be reacted with the C-terminal carboxyl group of each of the two peptide monomers.

(Addition of spacer)
The peptide compound of the present invention further comprises a spacer moiety. In one embodiment, the spacer may be incorporated into the peptide during peptide synthesis. For example, if the spacer contains one free amino group and a second functional group (eg, a carboxyl group or an amino group) that allows attachment to another molecular moiety, the spacer is attached to the solid support. Can be made.

In one embodiment, a spacer containing two functional groups is first coupled to the solid support through the first functional group. Then, lysine linker L _K moiety having two functional groups capable of serving as initiation sites for peptide synthesis and a third functional group that enables binding to another molecular moiety (e.g., a carboxyl group or an amino group) , To the spacer through the second functional group of the spacer and the third functional group of the linker. Thereafter, the variation of solid phase synthesis techniques, two peptide monomers may be synthesized directly onto two reactive nitrogen groups of the linker L _K moiety. For example, a spacer attached to a solid support having a free amine group may be reacted with a lysine linker through the free carboxyl group of the linker.

  In an alternative embodiment, the spacer can be attached to the peptide dimer after peptide synthesis. Such binding can be achieved by methods well established in the art. In one embodiment, the linker contains at least one functional group suitable for attachment to the target functional group of the synthesized peptide. For example, a spacer having a free amine group can be reacted with the C-terminal carboxyl group of the peptide. In another example, a linker having a free carboxyl group can be reacted with the free amine group of lysine amide.

(Binding of polyethylene glycol (PEG))
In recent years, water-soluble polymers such as polyethylene glycol (PEG) have been used for covalent modification of peptides of therapeutic and diagnostic importance. Such polymer attachment is believed to enhance biological activity, prolong blood circulation time, reduce immunogenicity, increase water solubility, and increase resistance to protease digestion. For example, therapeutic polypeptides such as interleukins (Knauf et al. (1988) J. Biol. Chem. 263; 15064; and Tsutsumi et al. (1995) J. Controlled Release 33: 447), interferons (Kita et al. (1990 ) Drug Des. Delivery 6: 157), catalase (Abuchowski et al. (1977) J. Biol. Chem. 252: 582), superoxide dismutase (Beauchamp et al. (1983) Anal. Biochem. 131: 25), and adenosine Covalent attachment of PEGs to deaminases (such as Chen et al. (1981) Biochim. Biophy. Acta 660: 293) extends their in vivo half-life and / or reduces their immunogenicity and antigenicity. Has been reported.

  The peptide compounds of the present invention may comprise a polyethylene glycol (PEG) moiety covalently linked to a branched tertiary amide linker or peptide dimer spacer through a carbamate bond or an amide bond. An example of a PEG used in the present invention is a linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20K) to about 40K (the term “about” is described in the preparation of PEG. Indicating that some molecules are larger than the reported molecular weight and some are smaller). Preferably, PEG has a molecular weight of about 30K to about 40K.

  Another example of a PEG used in the present invention is a linear PEG having a molecular weight of about 10K to about 60K (the term “about” means a molecule larger than the molecular weight described in the preparation of PEG. Some are small molecules). Preferably, the PEG has a molecular weight of about 20K to about 40K. More preferably, the PEG has a molecular weight of about 20K.

  An example of a method of PEG covalent bonding (PEGylation) will be described below. There is no intent to be limited by these exemplary descriptions. One skilled in the art will appreciate that a variety of methods for covalently attaching a wide range of PEGs are well established in the art. Thus, peptide compounds to which PEG is attached by any of a number of attachment methods known in the art are encompassed by the present invention.

For example, PEG can be covalently attached to the linker through a reactive group capable of attaching an activated PEG molecule (eg, a free amino or carboxyl group). Methoxylated PEG (mPEG) with different reactive moieties can be used to attach PEG molecules to amino groups. Such polymers include mPEG-succinimidyl succinate, mPEG-succinimidyl carbonate, mPEG-imidate, mPEG-4-nitrophenyl carbonate, and mPEG-cyanuric chloride. Similarly, a methoxylated PEG with a free amine group (mPEG-NH ₂ ) can be used to attach a PEG molecule to a carboxyl group.

In some embodiments, the linker or spacer contains a terminal amino group (ie, located at the end of the spacer). This terminal amino group can be reacted with a suitably activated PEG molecule (such as mPEG-para-nitrophenyl carbonate (mPEG-NPC)) to create a stable covalent carbamate linkage. Alternatively, this terminal amino group can be a suitably activated PEG molecule containing a reactive N-hydroxyl-succinimide (NHS) group (eg, mPEG-succinimidyl butyrate) to create a stable covalent carbamate linkage. Rate (mPEG-SBA) or mPEG-succinimidyl propionate (mPEG-SPA) and the like. In other embodiments, the linker reactive group contains a carboxyl group that can be activated under appropriate reaction conditions to form a covalent bond with an amine-containing PEG molecule. Suitable PEG molecules include mPEG-NH ₂ and suitable reaction conditions include carbodiimide mediated amide formation and the like.

EPO-R agonist activity assay (in vitro functional assay)
The in vitro competitive binding assay quantifies the competitiveness of a test peptide against EPO for binding to EPO-R. For example (eg, as described in US Pat. No. 5,773,569), the extracellular domain of human EPO-R (EPO-binding protein, EBP) can be produced recombinantly in E. coli, and the recombinant protein can be produced on a solid support (micro Titer plates or synthetic beads) (eg, Sulfolink beads from Pierce Chemical, Rockford, Ill.). The immobilized EBP is then incubated with labeled recombinant EPO, or labeled recombinant EPO and test peptide. For such experiments, serial dilutions of test peptides are employed. The assay point with no added test peptide defines the total amount of EPO binding to EBP. For the reaction containing the test peptide, the amount of bound EPO is quantified and expressed as a percentage of control binding (total amount = 100%). These values are plotted against peptide concentration. IC50 values are defined as the concentration of test peptide that reduces EPO binding to EBP by 50% (ie, 50% inhibition of EPO binding).

  Different in vitro competitive binding assays measure the light signal produced as a function of two adjacent beads, a bead bound to EPO and a bead bound to EPO-R. Bead proximity is created by the binding of EPO to EPO-R. A test peptide that competes with EPO for binding to EPO-R prevents this binding and causes a decrease in light emission. The concentration of test peptide that resulted in a 50% reduction in light emission is defined as the IC50 value.

  The peptides of the invention compete with EPO very efficiently for binding to EPO-R. This enhanced function is demonstrated by the ability to inhibit EPO binding at substantially lower concentrations of peptides (ie, having a very low IC50 value).

  The biological activity and potency of the monomeric or dimeric peptide EPO-R agonists of the present invention that specifically bind to the EPO receptor can be measured using in vitro cell-based functional assays.

  One assay is based on a mouse pre-B cell line expressing human EPO-R and further transfected with a fos promoter driven luciferase reporter gene construct. When exposed to EPO or another EPO-R agonist, such cells respond by synthesizing luciferase. Luciferase causes light emission when the substrate luciferin is added. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity. The activity of the test peptide is measured by adding a serial dilution of the test peptide to the cells and then incubating it for 4 hours. After incubation, luciferin substrate is added to the cells and light emission is measured. The concentration of the test peptide that produced half the maximum light emission is recorded as the EC50.

  The peptides of the present invention show a dramatically enhanced ability to promote EPO-R signaling dependent luciferase expression in this assay. This enhanced function is indicated by the ability to produce half the maximum value of luciferase activity (ie, having a very low EC50 value) at substantially lower concentrations of peptide. This assay is the preferred method for estimating the potency and activity of the EPO-R agonist peptides of the present invention.

  Another assay is FDC-P1 / ER cells (Dexter et al. (1980) J. Exp. Med. 152: 1036-1047), a well-characterized untransformed mouse bone marrow derived cell line. It can be carried out using one in which EPO-R is stably transfected. These cells show EPO-dependent proliferation.

In one such assay, cells grow to a half stationary density in the presence of the necessary growth factors (eg, as described in US Pat. No. 5,773,569). The cells are then washed in PBS and starved for 16-24 hours in whole medium without growth factors. After measuring cell viability (eg by trypan blue staining), a stock solution (in whole medium without growth factors) is made to give approximately 10 ⁵ cells per 50 μL. Serial dilutions of the peptide EPO-R agonist compounds to be tested (typically free liquid phase as opposed to phage bound or other bound or immobilized peptides) (Which is a peptide) is made in a 96-well tissue culture plate and the final volume is 50 μL per well. Cells (50 μL) are added to each well and the cells are incubated for 24-48 hours (at this point the negative control should be dead or dormant). Cell proliferation is then measured by techniques known in the art (eg MTT assay: measuring H ³ -thymidine incorporation as an indicator of cell proliferation) (Mosmann (1983) J. Immunol. Methods 65: 55-63). Peptides are evaluated both in cell lines that express EPO-R and in parental cell lines that do not. The concentration of test peptide required to produce half of the maximum cell growth is recorded as the EC50.

  The peptides of the present invention show a dramatically enhanced ability to promote EPO-dependent cell proliferation in this assay. This enhanced function is indicated by the ability to produce half of the maximum value of cell growth stimulating activity at substantially lower concentrations of peptides (ie having a very low EC50 value). This assay is the preferred method for estimating the potency and activity of the EPO-R agonist peptides of the present invention.

  In another assay, cells are grown to stationary phase in medium supplemented with EPO, harvested, and then cultured in EPO-free medium for an additional 18 hours. Cells are divided into three groups with equal cell density. One group adds no additional factors (negative control), one group has EPO (positive control), and the experimental group has the test peptide. The cultured cells are then harvested at various time points, fixed, and stained with a DNA-binding fluorescent dye (eg, propidium iodide or Hoechst staining, both available from Sigma). Next, fluorescence is measured using, for example, FACS Scan Flow cytometry. Next, the percentage of cells in each phase of the cell cycle can be measured using, for example, the SOBR model of CelIFIT software (Becton Dickinson). Cells treated with EPO or active peptide show a higher percentage of cells in S phase compared to the negative control group (measured by increased fluorescence as an indicator of increased DNA content).

  Using cell lines of FDCP-1 (see eg Dexter et al. (1980) J. Exp. Med. 152: 1036-1047) or TF-1 (Kitamura et al. (1989) Blood 73: 375-380) Similar assays can be performed. FDCP-1 is a growth factor-dependent mouse pluripotent early hematopoietic progenitor cell that proliferates but does not differentiate when supplemented with WEHI-3 conditioned medium (medium containing IL-3, ATCC number TIB-68) Is a stock. For such experiments, FDCP-1 cell lines are transfected with human or mouse EPO-R to prepare FDCP-1-hEPO-R or FDCP-1-mEPO-R cell lines, respectively. They can grow in the presence of EPO but do not differentiate. TF-1, an EPO-dependent cell line, can also be used to measure the effect of peptide EPO-R agonists on cell proliferation.

In yet another assay, H ³ -thymidine incorporation into spleen cells as described in Krystal (1983) Exp. Hematol 11: 649-660 is used to confirm the ability of the compounds of the invention to function as EPO agonists. A microassay based procedure can be employed. Briefly, B6C3F ₁ mice are injected daily with phenylhydrazine (60 mg / kg) for 2 days. On day 3, spleen cells are removed and proliferative capacity over 24 hours is confirmed using the MTT assay.

  Binding of EPO to EPO-R in erythropoietin-responsive cell lines induces tyrosine phosphorylation of both receptors and a number of intracellular proteins including Shc, vav and JAK2 kinases. Thus, another in vitro assay measures the ability of the peptides of the invention to induce tyrosine phosphorylation of EPO-R and downstream intracellular signal transducer proteins. The active peptides identified by the binding and proliferation assays described above cause nearly the same phosphorylation pattern as in EPO in erythropoietin-responsive cells. For this assay, FDC-P1 / ER cells (Dexter et al. (1980) J Exp Med 152: 1036-47) are maintained in medium supplemented with EPO and grown to stationary phase. These cells are then cultured for 24 hours in medium without EPO. A defined number of such cells are then incubated with the test peptide at 37 ° C. for about 10 minutes. Each assay is also performed on a control sample of cells with EPO. The treated cells are then collected by centrifugation, resuspended in SDS lysis buffer and subjected to SDS polyacrylamide gel electrophoresis. The electrophoretic protein in the gel is transferred to nitrocellulose and the phosphotyrosine containing protein on the blot is visualized by standard immunological techniques. For example, blots can be probed with anti-phosphotyrosine antibodies (eg, mouse anti-phosphotyrosine IgG from Upstate Biotechnology, Inc.), washed, and secondary antibodies (eg, from Kirkegaard & Perry Laboratories, Inc., Washington, DC). Peroxidase-labeled goat anti-mouse IgG). The phosphotyrosine-containing protein can then be visualized by standard techniques, including colorimetric analysis, chemiluminescence assay, fluorescence analysis. For example, chemiluminescent assays can be performed using the Amersham ECL Western blotting system.

  Another cell-based in vitro assay that can be used to assess the activity of the peptides of the present invention is a colony assay using mouse bone marrow or human peripheral blood cells. Mouse bone marrow can be obtained from mouse femur and human peripheral blood samples can be obtained from healthy donors. In the case of peripheral blood, it is first isolated from mononuclear cell blood, for example, by centrifugation through a Ficoll-Hypac gradient (Stem Cell Technologies, Inc., Vancouver, Canada). For this assay, nucleated cells are counted to establish the number and concentration of nucleated cells in the original sample. Place a defined number of cells on methylcellulose according to manufacturer's instructions (Stem Cell Technologies, Inc., Vancouver, Canada). The experimental group is treated with the test peptide, the positive control group is treated with EPO, and the negative control group does not perform any treatment. Then, after a defined incubation period (typically 10 and 18 days), score the number of growing colonies in each group. The active peptide will promote colony formation.

Other in vitro biological assays that can be used to demonstrate the activity of the compounds of the present invention are disclosed in the following literature: Greenberger et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2931- 2935 (EPO-dependent hematopoietic progenitor cell line); Quelle and Wojchowski (1991) J. Biol. Chem. 266: 609-614 (protein tyrosine phosphorylation in B6SUt. EP cells); Dusanter-Fourt et al. (1992) J Biol. Chem. 287: 10670-10678 (tyrosine phosphorylation of the EPO receptor in human EPO-reactive cells); Quelle et al. (1992) J. Biol. Chem. 267: 17055-17060 (cytosol in FDC-ER cells) Protein, tyrosine phosphorylation of pp100); Worthington et al. (1987) Exp. Hematol. 15: 85-92 (colorimetric analysis of hemoglobin); Kaiho and Miuno (1985) Anal. Biochem. 149: 117-120 (2, Detection of hemoglobin using 7-diaminofluorene); Patel et al. (1992) J. Biol. Chem. 267: 21300-21302 (expression of c-myb); Witthuhn et al. (1993) Cell 74: 227-236 (JAK2 association and tyrosine phosphorylation); Leonard et al. (1993) Blood 82: 1071-1079 (expression of GATA transcription factor); and Ando et al. (1993) Proc. Natl. .. Acad Sci USA 90: (adjusted in G ₁ transition by cycling D2 and D3) 9571-9575.

  It has been reported that using instruments designed by Molecular Devices, known as microphysiometers, to measure the effects of agonists and antagonists on various receptors works well. The basis of this instrument is the measurement of the change in the acidification rate of the extracellular medium in response to receptor activation.

(Functional assay in vivo)
One in vivo functional assay that can be used to assess the efficacy of test peptides is a bioassay of erythropoietic / exhypoxic mice. For this assay, alternating conditioning cycles are applied to mice for several days. In this cycle, periods of low pressure conditions and periods of ambient pressure conditions are alternately applied to the mice. The mice are then maintained at ambient pressure for 2-3 days before administering the test peptide. Test peptide samples, or EPO standards in the case of positive control mice, are injected subcutaneously into conditioned mice. Two days later, radiolabeled iron (eg, ⁵⁹ Fe) is administered, and a blood sample is taken two days after administration of the radiolabeled iron. The hematocrit value and radioactivity are then measured for each blood sample by standard techniques. Blood samples from mice injected with the active test peptide will exhibit more radioactivity (due to binding of Fe ⁵⁹ by erythrocyte hemoglobin) than mice not injected with the test peptide or EPO.

Another in vivo functional assay that can be used to assess the efficacy of test peptides is the reticulocyte assay. For this assay, either EPO or test peptide is injected subcutaneously into normal untreated mice for 3 consecutive days. On day 3, mice are also injected intraperitoneally with dextran iron. On day 5, a blood sample is taken from the mouse. The percentage of reticulocytes in the blood is determined by staining with thiazole orange and flow cytometer analysis (reticulocyte count program). In addition, the hematocrit value is measured manually. The corrected reticulocyte percentage is determined using the following formula:
% RETIC _{correction value (CORRECTED)} =% RETIC _{observation value (OBSERVED)} X (Hematocrit _{individual value (INDIVIDUAL)} / Hematocrit _{normal value (NORMAL)} )
Active test compounds will have higher levels of% RETIC _CORRECTED compared to mice that were not injected with the test peptide or EPO.

Use of EPO-R Agonist Peptides of the Invention The peptide compounds of the invention are useful in therapeutic methods and pharmaceutical manufacturing methods. The peptide compounds of the invention can be administered to warm-blooded animals, including humans, to stimulate erythropoiesis in the presence of circulating neutralizing anti-EPO antibodies in vivo. Anti-EPO antibodies do not neutralize the effects of the peptide compounds of the present invention. Anti-EPO antibodies are of the IgG1 or IgG4 subtype and target the protein portion of EPO. In other embodiments, the anti-EPO antibody is of the IgG2, IgG3, IgA1, IgA2, IgD, IgE or IgM subtype.

  The peptide compounds of the present invention can be administered to warm-blooded animals to treat or cure disorders characterized by anti-EPO antibodies. For example, the peptide compounds of the invention could be used in the treatment of anti-EPO antibodies related to PRCA. Types of PRCA include autoimmune PRCA, antibody-mediated PRCA, drug or drug-induced acute self-limited PRCA, acquired chronic erythroblastic fistula, and congenital erythroblastic fistula. The peptide compounds of the invention could also be used in the treatment or prevention of antibody-mediated anemia in patients suffering from erythropoiesis. The peptide compounds of the invention could also be used in the treatment or prevention of anti-EPO antibody mediated anemia in patients with erythroblastosis.

  The peptide compounds of the invention can be administered to warm-blooded animals to ameliorate or treat anemia in patients with a disorder characterized by the presence of anti-EPO antibodies. The peptide compounds of the invention can be administered to ameliorate anemia in warm-blooded animals suffering from anti-EPO antibody-mediated PRCA. The peptide compounds of the invention can be administered to ameliorate anemia in warm-blooded animals that have developed anti-EPO antibodies, are undergoing dialysis, and are pre-dialysis CDKs. The peptide compound of the present invention can be administered to a warm-blooded animal suffering from chemotherapy-induced anemia in which an anti-EPO antibody has been produced. The peptide compound of the present invention is a warm-blooded animal suffering from PRCA mediated by an anti-EPO antibody in order to recover hemoglobin to a target range of 10 to 13 g / dL (preferably target range is 11 to 12 g / dL) Can be administered. The peptide compounds of the present invention can be administered to warm-blooded animals suffering from PRCA mediated by anti-EPO antibodies to restore hemoglobin to levels higher than 11 g / dL.

  The peptide compounds of the present invention can be administered to warm-blooded animals to prevent or reduce the onset of disorders characterized by anti-EPO antibodies. For example, the peptide compounds of the invention could be used in the prevention of anti-EPO PRCA. The peptide compounds of the invention can be administered to prevent or reduce the onset of anemia in warm-blooded animals having a disorder characterized by the presence of anti-EPO antibodies. The peptide compounds of the invention can be administered to prevent or reduce the onset of anemia in warm-blooded animals suffering from PRCA mediated by anti-EPO antibodies. The peptide compounds of the invention can be administered to prevent or reduce the onset of anemia in warm-blooded animals that have developed anti-EPO antibodies, are undergoing dialysis, and are pre-dialysis CDKs. The peptide compounds of the invention can be administered to prevent or reduce the onset of anemia in warm-blooded animals undergoing chemotherapy. In one aspect of the invention, the peptide compound of the invention is a warm-blooded animal undergoing dialysis and pre-dialysis CDK or suffering from chemotherapy-induced anemia, but recombinant EPO therapy Can be administered to prevent or reduce the onset of anemia in warm-blooded animals that have not yet received anti-EPO antibodies. The peptide compounds of the invention can be administered to warm-blooded animals suffering from PRCA mediated by anti-EPO antibodies in order to maintain hemoglobin at levels higher than 11 g / dL.

  Thus, the present invention encompasses methods of treatment or prevention of disorders characterized by anti-EPO antibodies, which administer an amount of a peptide of the present invention sufficient to stimulate EPO-R. Thereby alleviating symptoms associated with the presence of anti-EPO antibodies in vivo.

Pharmaceutical Composition In another aspect of the present invention, a pharmaceutical composition of the above-mentioned EPO-R agonist peptide compound is provided. Conditions that are alleviated or regulated by administration of such compositions include those described above. Such pharmaceutical compositions can be oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (passively or using iontophoresis or electroporation). For transmucosal (nasal, vaginal, rectal, or sublingual) administration, or for the use of biodegradable inserts, formulated into dosage forms appropriate for each administration route be able to. In general, the present invention comprises a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier together with an effective amount of an EPO-R agonist peptide or derivative product of the present invention. Includes pharmaceutical compositions. Such compositions include: dilutions of various buffers (eg Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizers (eg Tween 20, Tween 80 , Polysorbate 80), antioxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, thimerazol, benzyl alcohol) and expansive substances (eg, lactose, mannitol); polymer compounds (polylactic acid, polyglycolic acid, etc.) ) Incorporation of the material into a microparticle preparation or liposome. Hyaluronic acid can also be used. Such compositions can affect the physical state, stability, release rate in vivo, and rate of clearance in vivo of the proteins and derivatives of the invention. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Publishing Co. (18042 Easton, Pa.)) Pp. 1435-1712 (incorporated herein by reference). The composition can be prepared in liquid form or in the form of a dry powder (eg lyophilized).

(Oral delivery)
This specification contemplates the use of oral solid dosage forms and their outline is described in Chapter 89 of Remington's Pharmaceutical Sciences, 18th Edition 1990 (Mack Publishing Co. (Easton, PA)). (Incorporated herein by reference). Solid dosage forms include: tablets; capsules; pills; troches or medicinal candy; cachets; pellets; powders; Encapsulation with liposomes or proteinoids can also be used to formulate the compositions of the present invention (eg, proteinoid microspheres reported in US Pat. No. 4,925,673). Encapsulation with liposomes can be used, and liposomes can be derivatized with various polymers (eg, US Pat. No. 5,013,556). Possible solid dosage forms for treatment are described in Marshall, K., Modern Pharmaceutics, GS Banker and CT Rhodes, Chapter 10, 1979, which is incorporated herein by reference. In general, the formulation includes an EPO-R agonist peptide (or a chemically modified form thereof) and inert ingredients that allow protection against the gastric environment and release of biologically active material in the intestine. .

  The use of liquid dosage forms for oral administration is also contemplated herein, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which include other ingredients including: Inert diluents; adjuvants such as wetting agents; emulsifiers and suspending agents; and sweeteners, flavoring agents and perfuming agents.

  The peptide can be chemically modified so that oral delivery of the derivative is efficacious. In general, a contemplated chemical modification is one that attaches at least one moiety to the component molecule itself, where this moiety is: (a) inhibition of proteolysis; and (b) from the stomach or intestine Allows uptake into the bloodstream. It is also desirable to increase the overall stability of the component (s) and increase the time it circulates in the body. As mentioned above, PEGylation is a preferred chemical modification for pharmaceutical use. Other moieties that can be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-1,3-dioxolane and poly-1 , 3,6-thioxocan (eg Abuchowski and Davis (1981), Enzymes as Drugs, "Soluble Polymer-Enzyme Adducts," edited by Hocenberg and Roberts (Wiley-Interscience, New York, NY) pp. 367-383; and Newmark et al., (1982) J. Appl. Biochem. 4: 185-189).

  For oral formulations, the location of release may be the stomach, small intestine (duodenum, jejunum or ileum), or large intestine. Those skilled in the art have available formulations that do not dissolve in the stomach but release the material in the duodenum or elsewhere in the intestine. Preferably, release, protection of the peptide (or derivative) or release of the peptide (or derivative) will avoid adverse effects of the stomach environment beyond the stomach environment (such as in the small intestine).

  In order to ensure complete stomach resistance, a coating that is impervious to at least pH 5.0 is essential. Examples of more common inert ingredients used as enteric coatings are: cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate ( PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings can be used as mixed thin films.

  The coating or mixture of coatings can also be used on tablets and is not intended for protection against the stomach. This includes sugar coatings or coatings that make it easier to swallow tablets. Capsules may consist of a hard shell (such as gelatin) for delivering a dry therapeutic agent (ie, a powder), and for liquid forms a soft gelatin shell can be used. The cachet shell material may be dark starch or other food paper. For pills, medicinal candy, molded tablets or powder tablets, techniques of massing can be used.

  The peptide (or derivative) can be included in the formulation as fine multiparticulates in the form of granules or pellets having a particle size of about 1 mm. The formulation of the material for capsule administration may be a powder, a lightly compressed plug, or a tablet. These therapeutic agents can be prepared by compression.

  Coloring and / or flavoring agents can also be included. For example, the peptide (or derivative) can be formulated (such as by encapsulation with liposomes or microspheres) and then further contained in a chilled beverage containing edible products such as colorants and flavoring agents.

  The volume of the peptide (or derivative) can be diluted or increased with an inert material. These diluents can include: carbohydrates (especially mannitol), alpha-lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch. Certain inorganic salts can also be used as fillers, including: calcium triphosphate, magnesium carbonate and sodium chloride. Commercially available diluents include Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

  A disintegrant can be included in formulating the therapeutic agent into a solid dosage form. Materials used as disintegrants include, but are not limited to, starch (including extract protab, which is a commercially available disintegrant based on starch). The following can all be used: sodium starch glycolate, amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge and bentonite. The disintegrant may be an insoluble cation exchange resin. Powdered rubbers can also be used as disintegrants and binders and can include powdered rubbers such as agar, karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

  Binders can be used together to retain the drug of the peptide (or derivative) and form a hard tablet, derived from natural products such as gum arabic, tragacanth, starch and gelatin Of materials included. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Both polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can be used in alcoholic solutions to granulate the peptide (or derivative).

  In order to prevent adhesion during the formulation process, anti-friction agents can be included in the formulation of the peptide (or derivative). Lubricants can be used as a layer between the peptide (or derivative) and the die wall, which can include but are not limited to: stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE ), Liquid paraffin, vegetable oils and waxes. Water-soluble lubricants (sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, carbowax 4000 and 6000, etc.) can also be used.

  Glidants can be added during formulation to improve the flow properties of the drug and to aid rearrangement during compression. Glidants can include starch, talc, calcined silica and hydrated silicoaluminate.

  A surfactant can be added as a wetting agent to aid dissolution of the peptide (or derivative) in an aqueous environment. Surfactants can include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents can also be used and can include benzalkonium chloride or benzethomium chloride. A list of potential anionic detergents that can be included in the formulation as surfactants is as follows: Lauromacrogol 400; polyoxyl 40 stearate; polyoxyethylene hydrogenated castor oil 10, 50 and 60; glycerol mono Stearates; polysorbates 20, 40, 60, 65 and 80; sucrose fatty acid esters; methylcellulose; and carboxymethylcellulose. These surfactants can be present in protein or derivative formulations alone or as a mixture in various proportions.

  Additives that potentially enhance uptake of peptides (or derivatives) are, for example, fatty acids, oleic acid, linoleic acid and linolenic acid.

  A controlled release oral formulation would be desirable. The peptide (or derivative) can be incorporated into an inert matrix (eg, rubber) that allows release by a diffusion or leaching mechanism. Slowly modifying matrices can also be incorporated into the formulation. Some enteric coatings also have the effect of delaying release. Another form of controlled release is by a method based on the Oros therapeutic system (Alza). That is, the drug is placed in a semipermeable membrane that passes water and pushes the drug out through a single small opening due to the effect of osmotic pressure.

  Other coatings can be used during formulation. These include a variety of sugars that can be applied in coating pans. Peptides (or derivatives) can also be provided in thin film coated tablets and the materials used in this case are divided into two groups. The first is a non-enteric material that includes: methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, providone and polyethylene glycol. The second group consists of enteric materials that are typically esters of phthalic acid.

  Mixtures of materials can be used to provide an optimal thin film coating. Thin film coating can be done in a pan coater or fluidized bed, or by compression coating.

(Parenteral delivery)
The preparation for parenteral administration of the present invention comprises a sterilized water-soluble or water-insoluble solution, suspension or emulsion. Examples of water-insoluble solvents or vehicles are: propylene glycol; polyethylene glycol; vegetable oils (such as olive oil and corn oil); gelatin; and injectable organic esters (such as ethyl oleate). Such dosage forms can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. They can be sterilized, for example, by filtration through a bacteria retaining filter, incorporation of a sterilant into the composition, irradiation of the composition, or heating of the composition. It can also be produced using sterile water or other sterile injectable media immediately prior to use.

(Rectal or vaginal delivery)
Compositions for rectal or vaginal administration are preferably suppositories that may contain, in addition to the active substance, excipients (such as cocoa butter or a suppository wax). Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.

(Pulmonary delivery)
The present specification also contemplates pulmonary delivery of EPO-R agonist peptides (or derivatives thereof). The peptide (or derivative) is delivered to the lungs of the mammal during inhalation and passes through the epithelial layer of the lungs to the bloodstream (see, eg, Adjei et al. (1990) Pharmaceutical Research 7: 565-569; Adjei et al. (1990) Int. J. Pharmaceutics 63: 135-144 (leuprolide acetate); Braquet et al. (1989) J. Cardiovascular Pharmacology 13 (sup5): 143-146 (endothelin-1); Hubbard et al. (1989) ) Annals of Internal Medicine, Vol. III, pp. 206-212 (α1-antitrypsin); Smith et al. (1989) J. Clin. Invest. 84: 1145-1146 (α-1-proteinase); Oswein et al. (1990) "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II Keystone, Colorado (recombinant human growth hormone); Debs et al. (1988) J. Immunol. 140: 3482-3488 (interferon-γ and tumor necrosis factor) α); and Platz et al., US Pat. No. 5,284,656 (granulocyte colony stimulating factor)). Methods and compositions for pulmonary delivery of drugs for systemic action are described in US Pat. No. 5,451,569 to Wong et al.

  A wide range of mechanical devices designed for pulmonary delivery of therapeutic agents are contemplated for use in the practice of the present invention, including nebulizers, metered dose inhalers, and powder inhalers. Without limitation, all of these are well known to those skilled in the art. Specific examples of commercially available devices suitable for the practice of the present invention include Ultravent nebulizers (Mallinckrodt (St. Louis, MO)), Acorn II nebulizers (Marquest Medical Products (Englewood, CO)), Ventolin metered dose inhalers (Glaxo (Research Triangle Park, North Carolina)), and Spinhaler powder inhalers (Fisons, Bedford, Mass.).

  All of these devices require the use of appropriate formulations for peptide (or derivative) dosing. Typically, each formulation is specific to the type of device employed and can involve the use of suitable propellant materials in addition to the usual diluents, adjuvants and / or carriers useful for therapy. Also contemplated are the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers. Depending on the type of chemical modification or the type of equipment employed, chemically modified peptides can also be prepared in different formulations.

  Formulations suitable for use with nebulizers, either jet or ultrasonic, are typically dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. Containing a peptide (or derivative). The formulation can also include a buffer and a simple sugar (eg, for protein stabilization and regulation of osmotic pressure). The nebulizer formulation can also contain a surfactant to reduce or prevent surface-induced aggregation of peptides (or derivatives) caused by spraying of the solution in the formation of the aerosol.

  Formulations for use with metered dose inhalers generally include a finely divided powder containing a peptide (or derivative) suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose. For example, chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons (including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, or combinations thereof). Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may also be useful as a surfactant.

  Formulations for dosing from powder inhalation devices include finely divided dry powders containing peptides (or derivatives), and can also include swelling agents (such as lactose, sorbitol, sucrose or mannitol) Is an amount that facilitates dispersion of the powder from the device (eg, 50-90 wt% of the formulation). The peptide (or derivative) should be most advantageously prepared in particulate form, with an average particle size of less than 10 mm (or microns), most preferably 0.5-5 mm, in order to deliver most effectively to the end of the lung It is.

(Nasal delivery)
Nasal delivery of the EPO-R agonist peptide (or derivative) is also contemplated. Nasal delivery allows the peptide to reach the bloodstream immediately after administration of the therapeutic agent to the nose, without having to deposit the therapeutic agent in the lungs. Formulations for nasal delivery include those having dextran or cyclodextran.

  It is also contemplated to use other transdermal absorption enhancers used to facilitate nasal delivery with the peptides of the present invention (WO 2004/056314 filed on Dec. 17, 2003). Which is incorporated herein by reference in its entirety).

(dose)
Further studies are performed on all peptide compounds to provide information on the appropriate doses for treating various conditions in various patients. Given the treatment status, age, and overall health status of the person receiving the medication, one skilled in the art will be able to determine the appropriate dosage. The dose selected will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. In general, mammals are administered daily doses of 0.001-10 mg / kg body weight. The dosing schedule depends on the half-life in the circulation and the formulation used.

  The therapeutic dosage range in the methods of the invention may be from 0.05 to 0.3 milligrams (mg) of compound (0.05 to 0.3 mg / kg) per kilogram (kg) of an individual's body weight. More specifically, the dosage range is preferably 0.05 to 0.2 mg / kg. In addition, the physician can start with a dose of 0.05 mg / kg and increase the dose by about 25-50% for each individual being treated based on the individual's hemoglobin response. In this way, the physician can increase the individual dose until an appropriate hemoglobin response is obtained. For individuals who are dialysis-inexperienced patients, dialysis patients, or patients with chemotherapy-induced anemia, an appropriate hemoglobin response is to increase the target level of about 11-12 g / dL of hemoglobin. You will get, or you will get another hemoglobin level as determined by your doctor.

  A number of administration routes can be used (as described above, oral administration, intravenous administration, etc.). The preferred route of administration will be subcutaneous. Another route of administration would be intravenous administration. Preferred compounds for use in the method of the present invention include those shown below.

Carbamate linkage; no sarcosine; PEG weight range (herein SEQ ID NO: 1):

Carbamate linkage; no sarcosine; preferred PEG weight (herein SEQ ID NO: 1):

Carbamate bond; with sarcosine; PEG weight range (here, SEQ ID NO: 2 is shown):

Carbamate linkage; with sarcosine; preferred PEG weight (herein SEQ ID NO: 2):

Amide bond; no sarcosine; PEG weight range (herein SEQ ID NO: 1):

Amide bond; no sarcosine; preferred PEG weight (herein SEQ ID NO: 1):

Amide bond; with sarcosine; PEG weight range (SEQ ID NO: 2 is shown here):

Amide bond; with sarcosine; preferred PEG weight (herein SEQ ID NO: 2):

  The peptides (or derivatives thereof) of the invention can be administered with one or more additional active ingredients or pharmaceutical compositions. For example, the peptides of the present invention can be administered with other peptide compounds that activate EPO-R, or with active ingredients or pharmaceutical compositions.

EXAMPLES Next, the present invention will be described based on the following examples. However, these examples and other examples described herein are provided by way of illustration only and are not intended to limit the scope and meaning of the present invention or any illustrated form. Likewise, the invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art after reading this specification and can be made without departing from the spirit and scope of the invention. Accordingly, the invention is to be limited only by the terms and terms in the appended claims, along with the full scope of content equivalent to that claimed.

Synthesis of EPO-R agonist peptide dimers by solid phase synthesis
Step 1—Synthesis of Cbz-TAP :
A solution containing commercially available diamine (“TAP” from Aldrich Chemical) (1Og, 67.47 mmol) in anhydrous DCM (100 ml) was cooled to 0 ° C. A solution of benzyl chloroformate (4.82 ml, 33.7 mmol) in anhydrous DCM (50 ml) was added slowly through a dropping funnel over 6-7 hours, keeping the temperature of the reaction mixture at 0 ° C. and room temperature. (Up to 25 ° C). After an additional 16 hours, DCM was removed under vacuum and the residue was partitioned between 3N HCl and ether. The aqueous layer was collected, neutralized to pH 8-9 with 50% aqueous NaOH, and extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ and then concentrated under vacuum to prepare crude mono-Cbz-TAP (5 g, about 50% yield). This compound was used in the next reaction without further purification.

Step 2—Synthesis of Cbz-TAP-Boc :
To a vigorously stirred suspension of Cbz-TAP (5 g, 17.7 mmol) in hexane (25 ml) was added Boc ₂ O (3.86 g, 17.7 mmol) and stirring was continued overnight at room temperature. The reaction mixture was diluted with DCM (25 ml) and washed with 10% aqueous citric acid (2 ×), water (2 ×) and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under vacuum. The crude product (yield 5 g) was used directly in the next reaction.

Step 3-Synthesis of Boc-TAP :
The crude product from the previous reaction was dissolved in methanol (25 ml) and hydrogenated for 16 hours under balloon pressure in the presence of 5% Pd on carbon (5% w / W). The mixture was filtered, washed with methanol, and the filtrate was concentrated in vacuo to prepare the crude H-TAP-Boc product (yield 3.7 g). The approximate total yield of Boc-TAP after completion of steps 1-3 was 44% (calculated based on the amount of Cbz-Cl used).

Step 4-Synthesis of TentaGel-linker :
TentaGel bromide (2.5 g, 0.48 mmol / g, Rapp Polymere (Germany)), phenolic acid linker (5-fold equivalent) and K ₂ CO ₃ (5-fold equivalent) in 20 mL of DMF for 14 hours, 70 Heated to ° C. After cooling to room temperature, the resin was washed (0.1 N HCl, water, ACN, DMF, MeOH) and dried to give an amber resin.

Step 5—Synthesis of TentaGel-Linker-TAP (Boc) :
2.5 gm of the above resin, H-TAP-Boc (1.5 gm, 5 eq) and glacial AcOH (34 μl, 5 eq) were placed in a 1: 1 ratio mixture of MeOH-THF and shaken overnight. . To this, 1M sodium cyanoborohydride solution in THF (5 times equivalent) was added and shaken for another 7 hours. The resin was filtered, washed (DMF, THF, 0.1 N HCl, water, MeOH) and dried. A small amount of resin was benzoylated with Bz-Cl and DIEA in DCM, cleaved with 70% TFA-DCM and checked by LCMS and HPLC.

Step 6—Synthesis of TentaGel-Linker-TAP-Lys :
The above resin is prepared by activating Fmoc-Lys (Fmoc) -OH solution (5 fold equivalents of amino acids and 5 fold equivalents of HATU at 0.5 M in DMF, followed by addition of 10 fold equivalents of DIEA ) And shake gently for 14 hours. The resin was washed (DMF, THF, DCM, MeOH) and dried to produce a protected resin. Residual amine groups were capped by treating the resin with a solution of 10% acetic anhydride and 20% pyridine in DCM for 20 minutes and then washed as described above. The Fmoc group was removed by gently shaking the resin for 20 minutes in 30% piperidine in DMF, then washed (DMF, THF, DCM, MeOH) and dried.

Step 7-TentaGel- linker -TAP-Lys (Peptide) ₂ Synthesis:
The above resin was applied to repeated cycles of Fmoc-amino acid binding with HBTU / HOBt activation, Fmoc was removed using piperidine, and both peptide chains were constructed simultaneously. This was conveniently performed using an ABI 433 automated peptide synthesizer available from Applied Biosystems. After the last Fmoc removal, the terminal amine group was acylated with acetic anhydride (10-fold equivalent) and DIEA (20-fold equivalent) in DMF for 20 minutes and then washed as above.

Step 8-Cutting from resin :
The above resin was suspended in a solution of TFA (82.5%), phenol (5%), ethanedithiol (2.5%), water (5%) and thioanisole (5%) for 3 hours at room temperature. Alternative cutting cocktails such as TFA (95%), water (2.5%) and triisopropylsilane (2.5%) can also be used. The TFA solution was cooled to 5 ° C. and poured into Et ₂ O to precipitate the peptide. Filtration and drying under reduced pressure gave the desired peptide. Pure peptide was obtained by purification by preparative HPLC using a C18 column.

Step 9-Oxidation of peptides to form intramolecular disulfide bonds :
The peptide dimer was dissolved in 20% DMSO / water (1 mg dry weight peptide / mL) and allowed to stand at room temperature for 36 hours. The peptide was purified by loading the reaction mixture onto a C18 HPLC column (Waters Delta-Pak C18, particle size 15 microns, pore size 300 angstrom, length 40 mm x 200 mm) followed by a linear gradient method (ACN / water / 0.01% TFA; ACN from 5% to 95%) was performed for 40 minutes. The fraction containing the desired peptide was lyopholized to give a fluffy white solid product.

Step 10— PEGylation of terminal-NH ₂ groups :
PEGylation through carbamate linkage :
The peptide dimer was mixed with 1.5 times equivalent (mole basis) activated PEG species (mPEG-NPC manufactured by NOF Corporation, Japan) in dry DMF to obtain a clear solution. After 5 minutes, 4-fold equivalent of DIEA was added to the above solution. The mixture was stirred at ambient temperature for 14 hours and then purified by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass spectrometry. The purified peptide was also subjected to purification through cation exchange chromatography as outlined below. The following scheme shows PEGylation of mPEG-NPC using SEQ ID NO: 3.

PEGylation through an amide bond :
The peptide dimer was mixed with 1.5 equivalents (molar basis) of 1 equivalent of activated PEG species (PEG-SPA-NHS from Shearwater, USA) in dry DMF to give a clear solution. After 5 minutes, 10-fold equivalent of DIEA was added to the above solution. The mixture was stirred at ambient temperature for 2 hours and then purified by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass spectrometry. The purified peptide was also subjected to purification through cation exchange chromatography as outlined below. The following scheme shows PEGylation of PEG-SPA-NHS using SEQ ID NO: 3.

Step 11-Purification by ion exchange :
Several exchange supports were investigated for their ability to separate the peptide-PEG bond from unreacted (or hydrolyzed) PEG in addition to the ability to retain the starting dimeric peptide. Ion exchange resin (2-3 g) was loaded onto a 1 cm column, then converted to the sodium form (0.2 N NaOH was loaded onto the column until the pH of the eluent was 14; about 5 column volumes), then hydrogen Convert to mold (eluted with either 0.1 N HCl or 0.1 M HOAc until eluent matched packing pH; approximately 5 column volumes), then wash with 25% ACN / water until pH 6 did. Peptide or peptide-PEG conjugate before conjugation was dissolved in 25% ACN / water (10 mg / mL), pH adjusted to less than 3 with TFA, and then loaded onto the column. After washing with 2-3 column volumes of 25% ACN / water and collecting 5 mL fractions, the peptide was released from the column by eluting with 25 M ACN / water 0.1 M NH ₄ OAc and again 5 mL fractions. Minutes were collected. Analysis by HPLC revealed which fractions contained the desired peptide. Analysis using an evaporative light scattering detector (ELSD) indicates that the peptide is not bound as a contaminant when the peptide is retained on the column and eluted with NH ₄ OAc solution (typically between fractions 4-10) Of PEG was not observed. When the peptide was eluted with the first wash buffer (generally the first two fractions), no desired PEG binding and excess PEG separation was observed.

  The following columns were successful in retaining both peptide and peptide-PEG linkages and were successful in purifying peptide-PEG linkages from unbound peptides.

Synthesis of EPO-R agonist peptide dimers by fragment condensation
Step 1: Synthesis of (Cbz) ₂ -Lys Under standard conditions, lysine is reacted with a benzyl chloroformate solution to give a lysine protected at its two amino groups with a Cbz group.

Step 2: Synthesis of Boc-TAP Boc-TAP is synthesized as described in Steps 1 to 3 of Example 1.

Step 3: Binding of (Cbz) ₂ -Lys and Boc-TAP Under standard binding conditions, (Cbz) ₂ -Lys and Boc-TAP are bound to obtain (Cbz) ₂ -Lys-TAP-Boc.

Process 4: Lys-TAP-Boc
The crude product from the previous reaction was dissolved in methanol (25 ml) and hydrogenated under balloon pressure for 16 hours in the presence of 5% Pd on carbon (5% w / W). The mixture was filtered and washed with methanol, and the filtrate was concentrated in vacuo to prepare the crude Lys-TAP-Boc product.

Step 5: Synthesis of peptide monomers by fragment condensation Four peptide fragments of peptide monomer sequences are synthesized by standard techniques. These partially protected fragments are then applied to two independent binding processes. In the first step, half of the N-terminal monomer is formed by linking two of the peptide fragments, while half of the C-terminal monomer is linking the other two peptide fragments. Formed by. In the second step of coupling, the N-terminal half and the C-terminal half are combined to form a fully protected monomer. The monomer is then deprotected with OBn by standard techniques.

Step 6: Oxidation of Peptide Monomers to Form Intramolecular Disulfide Bonds Next, OBn is deprotected and the condensed peptide monomer (SEQ ID NO: 12) is oxidized with iodide to protect cysteine protected with monomer Acm. Intramolecular disulfide bonds are formed between residues.

Step 7: Conjugation of Lys-TAP-Boc to oxidized, OBn-deprotected monomer to form peptide dimer Under standard conditions, Lys-TAP-Boc is oxidized in a 2-fold molar excess of OBn Binds to the deprotected monomer to form a peptide dimer. The peptide dimer is then deprotected under standard conditions.

Step 8: PEGylation of Deprotected Dimer Next, the deprotected peptide dimer is PEGylated as described in Step 10 of Example 1.

Step 9: Purification by Ion Exchange Next, the PEGylated peptide dimer is purified as described in Step 11 of Example 1.

In Vitro Activity Assays This example describes various in vitro assays useful in assessing the activity and efficacy of the EPO-R agonist peptides of the present invention. The results of these assays demonstrate that the novel peptides of the present invention bind to EPO-R and activate EPO-R signaling. Furthermore, the results of these assays show a surprising increase in EPO-R binding affinity and biological activity in the novel peptide composition compared to previously described EPO mimetic peptides.

EPO-R agonist peptide dimers are prepared according to the methods provided in Example 1 or 2. A series of in vitro activity assays (including reporter assays, proliferation assays, competitive binding assays, and C / BFU-e assays) are used to assess the potency of these peptide dimers. Further details on these four assays are given below.
The results of these in vitro activity assays are summarized in Table 2.

1. Reporter Assay This assay is based on a reporter cell from the mouse pre-B cell line, Baf3 / EpoR / GCSFR fos / lux. This reporter cell line expresses a chimeric receptor that includes from the extracellular portion of the human EPO receptor to the intracellular portion of the human GCSF receptor. This cell line is further transfected with a fos promoter driven luciferase reporter gene construct. As a result of activation of this chimeric receptor by the addition of an erythropoietic agent, the luciferase reporter gene is expressed. Thus, when luciferin, a luciferase substrate, is added, light is generated. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

Baf3 / EpoR / GCSFR fos / lux cells are 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (supernatant from culture of WEHI-3 cells; ATCC number TIB-68), And cultured in DMEM / F12 medium (Gibco) supplemented with penicillin / streptomycin. Approximately 18 hours prior to the assay, cells are starved by transferring them to DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day of the assay, cells were washed once with DMEM / F12 medium supplemented with 10% FBS (no WEHI-3 supernatant) and then supplemented with 10% FBS (no WEHI-3 supernatant) Cells in DMEM / F12 medium in the presence of known concentrations of test peptides or using EPO (R & D Systems (Minneapolis, MN)) as a positive control, cells (1 × 10 ⁶ cells / Incubate mL). In this assay, serial dilutions of test peptides are tested simultaneously. The assay plate is incubated for 4 hours at 37 ° C. in a 5% CO ₂ atmosphere, after which luciferin (Steady-Glo; Promega, Madison, Wis.) Is added to each well. After a 5 minute incubation, the light emission is measured using a Packard Topcount Luminometer (Packard Instrument, Downers Grove, Ill.). Light counts are plotted against test peptide concentration and analyzed using Graph Pad software. The concentration of the test peptide that results in a half-maximal emission of light is recorded as the EC50 (see Table 2; reporter EC50).

2. Proliferation assay This assay is based on the mouse pre-B cell line, Baf3, which is transfected to express human EPO-R. Growth of the resulting cell line, BaF3 / Gal4 / Elk / EPOR, depends on the activation of EPO-R. Quantify the degree of cell proliferation using MTT. At this time, the signal in the MTT assay is proportional to the number of viable cells.

Incubate BaF3 / Gal4 / Elk / EPOR cells in DMEM / F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC number TIB-68) in a spinner flask . Cultured cells are starved overnight at a cell density of 1 × 10 ⁶ cells / ml in DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant in a spinner flask. Next, the starved cells were washed twice with Dulbecco PBS (Gibco) and supplemented with 10% FBS (without WEHI-3 supernatant) in 1 × 10 ⁶ cells in DMEM / F12 medium Resuspend to a cell density of / ml. Next, 50 μL aliquots (˜50,000 cells) of cell suspension are plated in triplicate in 96 well assay plates. Serial dilutions of test EPO mimetic peptides in 50 μL aliquots in DMEM / F12 medium supplemented with 10% FBS (no WEHI-3 Supernatant I), or 50 μL EPO (R & D Systems, Minneapolis, MN) )) Or Aranesp ^™ (darbepoetin α; an EPO-R agonist commercially available from Amgen) is added to a 96-well assay plate (final well volume: 100 μL). For example, 12 different dilutions can be tested, with a final concentration of test peptide (or control EPO peptide) ranging from 81 OpM to 0.0045 pM. The plated cells are then incubated for 48 hours at 37 ° C. Next, 10 μL of MTT (Roche Diagnostics) is added to each culture plate well and incubated for 4 hours. The reaction is then stopped by adding 10% SDS + 0.01N HCl. The plate is then incubated overnight at 37 ° C. Next, the absorbance of each well at a wavelength of 595 nm is measured by spectrophotometry. A plot of absorbance readings against test peptide concentration is constructed and the EC50 is calculated using Graph Pad software. The concentration of the test peptide that results in an absorbance at half the maximum is recorded as the EC50 (see Table 2; proliferation EC50).

3. Competitive binding assay
Competitive binding using an assay that produces a light signal as a function of the proximity of two beads: a streptavidin donor bead with a biotinylated EPO-R binding peptide tracer and an acceptor bead to which EPO-R binds Perform the calculation. Light is produced by non-radiative energy transfer. When irradiated with light, singlet oxygen is released from the first bead, and contact with the released singlet oxygen causes light emission from the second bead. A set of these beads is commercially available (Packard). Bead proximity is created by the binding of EPO-R binding peptide tracer to EPO-R. For binding to EPO-R, a test peptide that competes with the EPO-R binding peptide tracer will prevent this binding and cause a decrease in light emission.

More specifically, the method is as follows. 4 μL of serial dilutions of test EPO-R agonist peptide, or positive or negative controls, are added to wells of a 384 well plate. Then add 2 μL / well of receptor / bead cocktail. The receptor bead cocktail consists of: 15 μL of 5 mg / ml streptavidin donor beads (Packard); 15 μL of 5 mg / ml monoclonal antibody abl79 (this antibody is a human placental alkaline phosphatase protein contained in recombinant EPO-R Recognize part); acceptor beads coated with protein A (protein A binds to abl79 antibody; Packard); 112.5 μL of recombinant EPO-R diluted 1: 6.6 (containing abl79 target epitope) For a portion of human placental alkaline phosphatase protein, produced as a fusion protein in Chinese hamster ovary cells); and 607.5 μL Alphaquest buffer (4OmM HEPES, pH 7.4; 1 mM MgCl ₂ ; 0.1% BSA, 0.05% Tween 20). Tap to mix. Add 2 μL / well of biotinylated EPO-R binding peptide tracer (final concentration: 3OnM). Using the sequence: biotin-GGLYACHMGPITWVCQPLRG (SEQ ID NO: 4), the peptide tracer EPO-R binding peptide (see “Receptor EC50 (pM)” in the table) is prepared according to the method described in Example 1.

  Centrifuge for 1 minute to mix. Seal the plate with a Packard Top Seal and wrap in foil. Incubate overnight at room temperature. After 18 hours, the light emission is read using an AlphaQuest reader (Packard). Plot light emission against peptide concentration and analyze with Graph Pad or Excel.

  The concentration of test peptide that resulted in a 50% decrease in light emission compared to the absence of test peptide is recorded as IC50 (see Table 2: AQ IC50).

4). C / BFU-e assay
EPO-R signaling stimulates bone marrow stem cells to differentiate and red blood cell progenitors to proliferate. This assay measures the ability of a test peptide to stimulate the proliferation and differentiation of erythroid progenitor cells from primary human bone marrow pluripotent stem cells.

For this assay, serial dilutions of test peptides are prepared in IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These serial dilutions, or positive control EPO peptide, are then added to methylcellulose to a final volume of 1.5 mL. The mixture of methylcellulose and peptide is then vortexed thoroughly. Thaw aliquots (100,000 cells / mL) of human bone marrow derived CD34 ⁺ cells (Poietics / Cambrex). In a 50 mL tube, gently add thawed cells to 0.1 mL lmg / ml DNAse (Stem Cells). Next, gently add 40-50 mL of IMDM medium to the cells. The medium is added drop-wise along the side of the 50 ml tube for the first 10 mL and the remaining medium is slowly dispensed along the side of the tube. The medium is then carefully removed by spinning the cells at 900 rpm for 20 minutes and gently aspirating. Resuspend cells in 1 ml IMDM medium and measure cell density per ml on hemacytometer slide (10 μL aliquot of cell suspension on slide; cell density is average number × 10,000 cells / mL) . Next, the cells are diluted with IMDM medium to a cell density of 15,000 cells / mL. Next, 100 μL of diluted cells is added to each 1.5 mL of methylcellulose and peptide sample (final cell concentration in assay medium is 1,000 cells / mL) and the mixture is vortexed. Remove bubbles in the mixture and aspirate 1 mL using a blunt needle. Add 0.25 mL of the aspirated mixture from each sample to each of the four wells of a 24-well plate (Falcon brand). The plated mixture is incubated for 14 days at 37 ° C. in 5% CO ₂ in a humid incubator. A phase contrast microscope (5-10x objective; final magnification 100x) is used to score for the presence of erythroid colonies. The test peptide concentration at which the number of colonies formed was 90% of the maximum compared to that observed with the EPO positive control is recorded as EC90 (see Table 2; C / BFU-e EC90).

5. Radioligand Competitive Binding Assay Alternatively, a radioligand competitive binding assay can be used to determine the IC ₅₀ value of the peptides of the present invention. This assay measures the binding of ¹²⁵ I-EPO to EPOr. The assay is preferably performed according to the following typical protocol.

B. Determination of appropriate receptor concentration One 50 μg vial of lyophilized recombinant EPOr extracellular domain fused to the Fc portion of human IgG1 is returned to 1 mL of assay buffer. To measure the correct amount of receptor for use in the assay, a 100 μL serial dilution of this receptor preparation was added with 200 μL of iodinated recombinant human erythropoietin ( ¹²⁵ I-EPO) in a 12 × 75 mm polypropylene tube. Bind at about 20,000cpm. Cap the tube and mix gently at 4 ° C overnight on a LabQuake rotary shaker.

  The next day, 50 μL of a 50% slurry of protein-G sepharose is added to each tube. Then incubate the tube for 2 hours at 4 ° C with gentle mixing. The tube is then centrifuged at 4000 RPM (3297 × G) for 15 minutes to pellet the protein-G sepharose. Carefully remove and discard the supernatant. After washing 3 times with 1 mL of 4 ° C assay buffer, the pellet is counted with a Wallac Wizard gamma counter. The results are then analyzed and the dilution required to reach 50% of the maximum binding is calculated.

C. Measuring IC ₅₀ for peptides To measure IC ₅₀ for peptide I, 100 μL of serial dilutions of peptide are combined with 100 μL of recombinant erythropoietin receptor (100 pg / tube) in a 12 × 75 mm polypropylene tube. . Next, 100 μL of iodinated recombinant human erythropoietin ( ¹²⁵ I-EPO) is added to each tube, and the tubes are capped and gently agitated at 4 ° C. overnight.

The next day, to quantify the ¹²⁵ I-EPO coupled as described above. The results are analyzed and IC ₅₀ values are calculated using Graphpad Prism version 4.0 from GraphPad Software (San Diego, Calif.). The assay is repeated two or more times for each peptide to be tested and repeated a total of three or more times to obtain an IC ₅₀ value measurement.

In Vivo Activity Assays This example describes various in vivo assays useful in assessing the activity and efficacy of the EPO-R agonist peptides of the present invention. EPO-R agonist peptide dimers are prepared according to the method provided in Example 1 or 2. The in vivo activity of these peptide monomers and dimers is assessed using serial assays, including erythrocytosis / exhypoxic mouse bioassays and reticulocyte assays. Further details on these two assays are given below.

1. Bioassay of erythrocytosis / hypoxia mice (Exhypoxic)
The test peptides are assayed for in vivo activity in a bioassay of erythropoietic and exhypoxic mice to which the method described in Cotes and Bangham (1961), Nature 191: 1065-1067 has been applied. In this assay, the test peptide functions as an EPO mimetic, ie, tests the ability to activate EPO-R and induce new erythrocyte synthesis. Erythrocyte synthesis is quantified based on the incorporation of radiolabeled iron into the hemoglobin of synthesized erythrocytes.

  BDF1 mice are acclimated to ambient conditions for 7-10 days. Weigh all animals and do not use low-weight animals (<15 grams). Mice are applied to a continuous conditioning cycle in a low pressure chamber for a total of 14 days. Each 24-hour cycle consists of 18 hours of 0.40 ± 0.02% atmospheric pressure and 6 hours of ambient pressure. After conditioning, the mice are maintained at ambient pressure for an additional 72 hours prior to dosing.

  Test peptides or recombinant human EPO standards are diluted in PBS + 0.1% BSA vehicle (PBS / BSA). The peptide monomer stock solution is first solubilized in dimethyl sulfoxide (DMSO). The negative control group includes one group of mice injected with PBS / BSA alone and one group of mice injected with 1% DMSO. Each dosing group includes 10 mice. Mice are injected subcutaneously (neck) with 0.5 mL of the appropriate sample.

48 hours after sample injection, mice are injected intraperitoneally with 0.2 ml of Fe ⁵⁹ (Dupont, NEN) (dose: approximately 0.75 μ Curie per mouse). 24 hours after administration of Fe ^59, the mice were weighed, 48 hours after administration of the Fe ^59, mice are sacrificed. Blood is collected from each animal by cardiac puncture and the hematocrit value is measured (heparin was used as an anticoagulant). Each blood sample (0.2 ml) is analyzed for Fe ⁵⁹ uptake using a Packard gamma counter. Unresponsive mice (ie, mice with less radioactive uptake than the negative control group) are excluded from the appropriate data set. Mice with a hematocrit value less than 53% of the negative control group are also excluded.

  Results are obtained from a set of 10 animals for each experimental dose. Calculate the average amount of radioactivity (counts per minute (CPM)) incorporated into blood samples obtained from each group.

2. Reticulocyte Assay Normal BDF1 mice receive either EPO control or test peptide for 3 consecutive days (dose: 0.5 mL, subcutaneous injection). On day 3, iron dextran (100 mg / ml) is also administered to the mice (dose: 0.1 mL, intraperitoneal injection). On day 5, mice are anesthetized with CO ₂ and bled by cardiac puncture. The percentage of reticulocytes in each blood sample is determined by staining with thiazole orange and flow cytometry analysis (reticulocyte count program). Measure hematocrit manually. The percentage of reticulocytes modified is determined using the following formula:
% RETIC _{correction value (CORRECTED)} =% RETIC _{observation value (OBSERVED)} X (Hematocrit _{individual value (INDIVIDUAL)} / Hematocrit _{normal value (NORMAL)} )

3. Hematology Assay Normal CD1 mice are administered either an EPO positive control, a test peptide, or a vehicle by intravenous bolus injection for 4 weeks. Positive control and test peptide dose ranges (shown in mg / kg) are tested by varying the active compound concentration in the formulation. The amount to be injected is 5 mg / kg. The vehicle control group consists of 12 animals, and each remaining dose group consists of 8 animals. Record daily viability and weekly body weight.

  The administered mice are fasted and anesthetized by isoflurane inhalation on day 1 (vehicle control mice) and on days 15 and 29 (4 animals / group / day), and terminal blood is obtained by cardiac puncture or abdominal aortic puncture. Collect the sample. Transfer blood to Vacutainer® brand tubes. A preferred anticoagulant is ethylenediaminetetraacetic acid (EDTA).

  Blood samples are evaluated using automated clinical analyzers (such as those manufactured by Coulter, Inc.) well known in the art, such as endpoints for measuring red blood cell synthesis and physiology (hematocrit (Hct), hemoglobin (Hgb) and total red blood cells. Number (RBC) etc.).

Synthesis of EPO-R agonist peptide homodimer of peptide monomer having amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1)
Step 1: Synthesis of Peptide Monomer Peptide monomers were prepared using standard Fmoc chemistry using TG-RAM resin (0.18 mmol / g, Rapp Polymere (Germany)) on an ABI 431A peptide synthesizer. To synthesize. For the synthesis of peptide monomers with an amidated carboxy terminus, the fully constructed peptide is cleaved from the resin using 82.5% TFA, 5% water, 6.25% anisole, 6.25% ethanedithiol. The deprotected product is filtered from the resin and precipitated with diethyl ether. After complete drying, the product is purified by C18 reverse phase high performance liquid chromatography using a gradient of acetonitrile / water in 0.1% trifluoroacetic acid. The structure of the peptide is confirmed by electrospray mass spectrometry. The peptide is dissolved in a 1: 1 solution of DMSO: water at a concentration of 1 mg / mL to cause disulfide formation. The product is purified by C18 reverse phase high performance liquid chromatography using a gradient of acetonitrile / water in 0.1% trifluoroacetic acid. Peptide monomers can be shown as follows:

Step 2: Synthesis of trifunctional linker
To a solution of diethyliminoacetate (10.Og, 52.8 mmol) and Boc-beta-alanine (10.Og, 52.8 mmol) in 100 mL DCM was added diisopropylcarbodiimide (8.0 mL, 51.1 mmol) at room temperature over 10 minutes. Added in. During the addition, the reaction mixture was warmed to 10 degrees, cooled to room temperature over 20 minutes. The reaction mixture was stirred overnight and the precipitated diisopropylurea was filtered. The solvent was removed under reduced pressure to give a gum, the residue was dissolved in ethyl acetate and filtered again to remove further precipitated urea. The organic phase is placed in a separatory funnel, washed (saturated NaHCO ₃ , brine, 0.5N HCl, brine), dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give the diester product as a colorless oil. Got. The diester was mixed with a 1: 1 mixture of MeOH: THF (100 mL) to which water (25 mL) was added followed by NaOH (5 g, 125 mmol). The pH was measured to be> 10. The reaction mixture was stirred at room temperature for 2 hours and acidified to pH 1 with 6N HCl. The aqueous phase was saturated with NaCl and extracted four times with ethyl acetate. The combined organic phase was washed (brine), dried (MgSO ₄ ) and concentrated under reduced pressure to give a white semi-solid. The solid was dissolved in 50 mL DCM and 300 mL hexane was added to make a white slurry. The solvent was removed under reduced pressure to give the dibasic acid as a white solid (14.7 g, 91.5% yield over 2 steps). N-hydroxysuccinimide (770 mg, 6.69 mmol), diisopropylcarbodiimide (1.00 mL, 6.38 mmol) and 4-dimethylaminopyridine (3 mg, 0.02 mmol) against a solution of dibasic acid (1 g, 3.29 mmol) in 20 mL DMF ) Was added. The reaction mixture was stirred overnight and the solvent was removed under reduced pressure. The residue was mixed with ethyl acetate and filtered to remove the precipitated urea. The organic phase is placed in a separatory funnel, washed (saturated NaHCO ₃ , brine, 0.5N HCl, brine), dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give di- The NHS ester product (1.12 g, 68% yield) was obtained.

Step 3: Binding of trifunctional linker to peptide monomer For linker binding , 2 equivalents of peptide were mixed with 1 equivalent of trifunctional linker in dry DMF to obtain a clear solution, 2 minutes Later add 5 times equivalent of DIEA. The mixture is stirred at ambient temperature for 14 hours. The solvent is removed under reduced pressure and the crude product is dissolved in 80% TFA in DCM for 30 minutes to remove the Boc group and then purified by C18 reverse phase HPLC. The structure of the dimer is confirmed by electrospray mass spectrometry. By this coupling reaction, the linker is coupled to the nitrogen atom of the ε amino group of the lysine residue of each monomer. The process using SEQ ID NO: 1 is shown below.

Step 4: PEGylation of peptide dimer
In PEGylated dry DMF through carbamate linkage , the peptide dimer is mixed with an equivalent (molar basis) of activated PEG species (mPEG-NPC from NOF Corporation, Japan) to obtain a clear solution. After 5 minutes, add 4 times equal amount of DIEA to this solution. The mixture is stirred at ambient temperature for 14 hours and then purified by C18 reverse phase HPLC. The structure of the PEGylated peptide is confirmed by MALDI mass spectrometry. The purified peptide was also applied for purification through cation exchange chromatography as outlined below. PEGylation of mPEG-NPC using SEQ ID NO: 1 is shown below.

In PEGylated dry DMF through amide bonds , the peptide dimer is mixed with an equivalent (molar basis) of activated PEG species (PEG-SPA-NHS from Shearwater, USA) to obtain a clear solution. After 5 minutes, add 10-fold equivalent of DIEA to this solution. The mixture is stirred at ambient temperature for 2 hours and then purified by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass spectrometry. The purified peptide was also applied for purification through cation exchange chromatography as outlined below. The PEGylation of PEG-SPA-NHS using SEQ ID NO: 1 is shown below.

Step 5: Purification by peptide ion exchange For some exchange supports, in addition to the ability to retain the starting dimeric peptide, the above peptide-PEG bond is separated from unreacted (or hydrolyzed) PEG. Investigate the ability to. Ion exchange resin (2-3 g) was loaded onto a 1 cm column, then converted to the sodium form (0.2 N NaOH was loaded onto the column until the pH of the eluent was 14; about 5 column volumes), then hydrogen Convert to mold (eluted with either 0.1 N HCl or 0.1 M HOAc until eluent matched packing pH; approximately 5 column volumes), then wash with 25% ACN / water until pH 6 did. Peptide or peptide-PEG conjugate before conjugation was dissolved in 25% ACN / water (10 mg / mL), pH adjusted to less than 3 with TFA, and then loaded onto the column. After washing with 2-3 column volumes of 25% ACN / water and collecting 5 mL fractions, the peptide was released from the column by eluting with 0.1 M NH ₄ OAc in 25% ACN / water and again 5 mL Fractions were collected. Analysis by HPLC revealed which fractions contained the desired peptide. Analysis using an evaporative light scattering detector (ELSD) indicates that the peptide is not bound as a contaminant when the peptide is retained on the column and eluted with NH ₄ OAc solution (typically between fractions 4-10) Of PEG was not observed. When the peptide was eluted with the first wash buffer (generally the first two fractions), no desired PEG binding and excess PEG separation was observed.

  The following columns were successful in retaining both peptide and peptide-PEG linkages and were successful in purifying peptide-PEG linkages from unbound peptides.

In the synthesis step 1 of the EPO-R agonist peptide homodimer of the peptide monomer having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), the peptide monomer synthesized is:
(AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2)
Except that it is an EPO-R agonist of a peptide monomer having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2) as described in Example 1. Synthesize peptide homodimers.

  When PEG is attached to the linker through a carbamate linkage, the final product of this synthesis using SEQ ID NO: 2 can be structurally shown as follows:

  When PEG is attached to the linker through an amide bond, the final product of this synthesis using SEQ ID NO: 2 can be shown structurally as follows:

In Vitro Activity Assays This example describes various in vitro assays useful in assessing the activity and efficacy of the EPO-R agonist peptides of the present invention. The results of these assays demonstrate that the novel peptides of the present invention bind to EPO-R and activate EPO-R signaling. Furthermore, the results of these assays show a surprising increase in EPO-R binding affinity and biological activity in the novel peptide composition compared to previously described EPO mimetic peptides.

EPO-R agonist peptide monomers and dimers are prepared according to the methods provided in Example 1 or 2. A series of in vitro activity assays (including reporter assays, proliferation assays, competitive binding assays, and C / BFU-e assays) are used to assess the potency of these peptide dimers. Further details on these four assays are given below.
The results of these in vitro activity assays are summarized in Table 2.

1. Reporter Assay This assay is based on a reporter cell from the mouse pre-B cell line, Baf3 / EpoR / GCSFR fos / lux. This reporter cell line expresses a chimeric receptor that includes from the extracellular portion of the human EPO receptor to the intracellular portion of the human GCSF receptor. This cell line is further transfected with a fos promoter driven luciferase reporter gene construct. As a result of activation of this chimeric receptor by the addition of an erythropoietic agent, the luciferase reporter gene is expressed. Thus, when luciferin, a luciferase substrate, is added, light is generated. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

Baf3 / EpoR / GCSFR fos / lux cells are 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (supernatant from culture of WEHI-3 cells; ATCC number TIB-68), And cultured in DMEM / F12 medium (Gibco) supplemented with penicillin / streptomycin. Approximately 18 hours prior to the assay, cells are starved by transferring them to DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day of the assay, cells were washed once with DMEM / F12 medium supplemented with 10% FBS (no WEHI-3 supernatant) and then supplemented with 10% FBS (no WEHI-3 supernatant) Cells in DMEM / F12 medium in the presence of known concentrations of test peptides or using EPO (R & D Systems (Minneapolis, MN)) as a positive control, cells (1 × 10 ⁶ cells / Incubate mL). In this assay, serial dilutions of test peptides are tested simultaneously. The assay plate is incubated for 4 hours at 37 ° C. in a 5% CO ₂ atmosphere, after which luciferin (Steady-Glo; Promega, Madison, Wis.) Is added to each well. After a 5 minute incubation, the light emission is measured using a Packard Topcount Luminometer (Packard Instrument, Downers Grove, Ill.). Light counts are plotted against test peptide concentration and analyzed using Graph Pad software. The concentration of test peptide that results in a half-maximal light emission is recorded as the EC50.

2. Proliferation assay This assay is based on the mouse pre-B cell line, Baf3, which is transfected to express human EPO-R. Growth of the resulting cell line, BaF3 / Gal4 / Elk / EPOR, depends on the activation of EPO-R. Quantify the degree of cell proliferation using MTT. At this time, the signal in the MTT assay is proportional to the number of viable cells.

Incubate BaF3 / Gal4 / Elk / EPOR cells in DMEM / F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC number TIB-68) in a spinner flask . Cultured cells are starved overnight at a cell density of 1 × 10 ⁶ cells / ml in DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant in a spinner flask. Next, the starved cells were washed twice with Dulbecco PBS (Gibco) and supplemented with 10% FBS (without WEHI-3 supernatant) in 1 × 10 ⁶ cells in DMEM / F12 medium Resuspend to a cell density of / ml. Next, 50 μL aliquots (˜50,000 cells) of cell suspension are plated in triplicate in 96 well assay plates. Serial dilutions of test EPO mimetic peptides in 50 μL aliquots in DMEM / F12 medium supplemented with 10% FBS (no WEHI-3 Supernatant I), or 50 μL EPO (R & D Systems, Minneapolis, MN) )) Or Aranesp ^™ (darbepoetin α; an EPO-R agonist commercially available from Amgen) is added to a 96-well assay plate (final well volume: 100 μL). For example, 12 different dilutions can be tested, with a final concentration of test peptide (or control EPO peptide) ranging from 81 OpM to 0.0045 pM. The plated cells are then incubated for 48 hours at 37 ° C. Next, 10 μL of MTT (Roche Diagnostics) is added to the wells of each culture plate and then incubated for 4 hours. The reaction is then stopped by adding 10% SDS + 0.01N HCl. The plate is then incubated overnight at 37 ° C. Next, the absorbance of each well at a wavelength of 595 nm is measured by spectrophotometry. A plot of absorbance readings against test peptide concentration is constructed and the EC50 is calculated using Graph Pad software. The test peptide concentration at which the absorbance is half the maximum is recorded as the EC50.

3. Competitive binding assay
Competitive binding using an assay that produces a light signal as a function of the proximity of two beads: a streptavidin donor bead with a biotinylated EPO-R binding peptide tracer and an acceptor bead to which EPO-R binds Perform the calculation. Light is produced by non-radiative energy transfer. When irradiated with light, singlet oxygen is released from the first bead, and contact with the released singlet oxygen causes light emission from the second bead. A set of these beads is commercially available (Packard). Bead proximity is created by the binding of EPO-R binding peptide tracer to EPO-R. For binding to EPO-R, a test peptide that competes with the EPO-R binding peptide tracer will prevent this binding and cause a decrease in light emission.

More specifically, the method is as follows. 4 μL of serial dilutions of test EPO-R agonist peptide, or positive or negative controls, are added to wells of a 384 well plate. Then add 2 μL / well of receptor / bead cocktail. The receptor bead cocktail consists of: 15 μL of 5 mg / ml streptavidin donor beads (Packard); 15 μL of 5 mg / ml monoclonal antibody abl79 (this antibody is a human placental alkaline phosphatase protein contained in recombinant EPO-R Recognize part); acceptor beads coated with protein A (protein A binds to abl79 antibody; Packard); 112.5 μL of recombinant EPO-R diluted 1: 6.6 (containing abl79 target epitope) For a portion of human placental alkaline phosphatase protein, produced as a fusion protein in Chinese hamster ovary cells); and 607.5 μL Alphaquest buffer (4OmM HEPES, pH 7.4; 1 mM MgCl ₂ ; 0.1% BSA, 0.05% Tween 20). Tap to mix. Add 2 μL / well of biotinylated EPO-R binding peptide tracer (final concentration: 3OnM). Using SEQ ID NO: 4, the peptide tracer EPO-R binding peptide (see “Receptor EC50 (pM)” in the table) is prepared according to the method described in Example 1.

  Centrifuge for 1 minute to mix. Seal the plate with a Packard Top Seal and wrap in foil. Incubate overnight at room temperature. After 18 hours, the light emission is read using an AlphaQuest reader (Packard). Plot light emission against peptide concentration and analyze with Graph Pad or Excel.

  The concentration of test peptide that resulted in a 50% decrease in light emission compared to the absence of test peptide is recorded as the IC50.

4). C / BFU-e assay
EPO-R signaling stimulates bone marrow stem cells to differentiate and red blood cell progenitors to proliferate. This assay measures the ability of a test peptide to stimulate the proliferation and differentiation of erythroid progenitor cells from primary human bone marrow pluripotent stem cells.

For this assay, serial dilutions of test peptides are prepared in IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These serial dilutions, or positive control EPO peptide, are then added to methylcellulose to a final volume of 1.5 mL. The mixture of methylcellulose and peptide is then vortexed thoroughly. Thaw aliquots (100,000 cells / mL) of human bone marrow derived CD34 ⁺ cells (Poietics / Cambrex). In a 50 mL tube, gently add thawed cells to 0.1 mL lmg / ml DNAse (Stem Cells). Next, gently add 40-50 mL of IMDM medium to the cells. The medium is added drop-wise along the side of the 50 ml tube for the first 10 mL and the remaining medium is slowly dispensed along the side of the tube. The medium is then carefully removed by spinning the cells at 900 rpm for 20 minutes and gently aspirating. Resuspend cells in 1 ml IMDM medium and measure cell density per ml on hemacytometer slide (10 μL aliquot of cell suspension on slide; cell density is average number × 10,000 cells / mL) . Next, the cells are diluted with IMDM medium to a cell density of 15,000 cells / mL. Next, 100 μL of diluted cells is added to each 1.5 mL of methylcellulose and peptide sample (final cell concentration in assay medium is 1,000 cells / mL) and the mixture is vortexed. Remove bubbles in the mixture and aspirate 1 mL using a blunt needle. Add 0.25 mL of the aspirated mixture from each sample to each of the four wells of a 24-well plate (Falcon brand). The plated mixture is incubated for 14 days at 37 ° C. in 5% CO ₂ in a humid incubator. A phase contrast microscope (5-10x objective; final magnification 100x) is used to score for the presence of erythroid colonies. The test peptide concentration at which the number of colonies formed was 90% of the maximum compared to that observed with the EPO positive control is recorded as EC90 (see Table 2; C / BFU-e EC90).

In Vivo Activity Assays This example describes various in vivo assays useful in assessing the activity and efficacy of the EPO-R agonist peptides of the present invention. EPO-R agonist peptide monomers and dimers are prepared according to the method provided in Example 1. The in vivo activity of these peptide monomers and dimers is assessed using serial assays, including erythrocytosis / exhypoxic mouse bioassays and reticulocyte assays. Further details on these two assays are given below.

1. Bioassay of erythrocytosis / hypoxia mice (Exhypoxic)
The test peptides are assayed for in vivo activity in a bioassay of erythropoietic and exhypoxic mice to which the method described in Cotes and Bangham (1961), Nature 191: 1065-1067 has been applied. In this assay, the test peptide functions as an EPO mimetic, ie, tests the ability to activate EPO-R and induce new erythrocyte synthesis. Erythrocyte synthesis is quantified based on the incorporation of radiolabeled iron into the hemoglobin of synthesized erythrocytes.

  BDF1 mice are acclimated to ambient conditions for 7-10 days. Weigh all animals and do not use low-weight animals (<15 grams). Mice are applied to a continuous conditioning cycle in a low pressure chamber for a total of 14 days. Each 24-hour cycle consists of 18 hours of 0.40 ± 0.02% atmospheric pressure and 6 hours of ambient pressure. After conditioning, the mice are maintained at ambient pressure for an additional 72 hours prior to dosing.

  Test peptides or recombinant human EPO standards are diluted in PBS + 0.1% BSA vehicle (PBS / BSA). The peptide monomer stock solution is first solubilized in dimethyl sulfoxide (DMSO). The negative control group includes one group of mice injected with PBS / BSA alone and one group of mice injected with 1% DMSO. Each dosing group includes 10 mice. Mice are injected subcutaneously (neck) with 0.5 mL of the appropriate sample.

48 hours after sample injection, mice are injected intraperitoneally with 0.2 ml of Fe ⁵⁹ (Dupont, NEN) (dose: approximately 0.75 μ Curie per mouse). 24 hours after administration of Fe ^59, the mice were weighed, 48 hours after administration of the Fe ^59, mice are sacrificed. Blood is collected from each animal by cardiac puncture and the hematocrit value is measured (heparin was used as an anticoagulant). Each blood sample (0.2 ml) is analyzed for Fe ⁵⁹ uptake using a Packard gamma counter. Unresponsive mice (ie, mice with less radioactive uptake than the negative control group) are excluded from the appropriate data set. Mice with a hematocrit value less than 53% of the negative control group are also excluded.

  Results are obtained from a set of 10 animals for each experimental dose. Calculate the average amount of radioactivity (counts per minute (CPM)) incorporated into blood samples obtained from each group.

2. Reticulocyte Assay Normal BDF1 mice receive either EPO control or test peptide for 3 consecutive days (dose: 0.5 mL, subcutaneous injection). On day 3, iron dextran (100 mg / ml) is also administered to the mice (dose: 0.1 mL, intraperitoneal injection). On day 5, mice are anesthetized with CO ₂ and bled by cardiac puncture. The percentage of reticulocytes in each blood sample is determined by staining with thiazole orange and flow cytometry analysis (reticulocyte count program). Measure hematocrit manually. The percentage of reticulocytes modified is determined using the following formula:
% RETIC _{correction value (CORRECTED)} =% RETIC _{observation value (OBSERVED)} X (Hematocrit _{individual value (INDIVIDUAL)} / Hematocrit _{normal value (NORMAL)} )

3. Hematology Assay Normal CD1 mice are administered either an EPO positive control, a test peptide, or a vehicle by intravenous bolus injection for 4 weeks. Positive control and test peptide dose ranges (shown in mg / kg) are tested by varying the active compound concentration in the formulation. The amount to be injected is 5 mg / kg. The vehicle control group consists of 12 animals, and each remaining dose group consists of 8 animals. Record daily viability and weekly body weight.

  The administered mice are fasted and anesthetized by isoflurane inhalation on day 1 (vehicle control mice) and on days 15 and 29 (4 animals / group / day), and terminal blood is obtained by cardiac puncture or abdominal aortic puncture. Collect the sample. Transfer blood to Vacutainer® brand tubes. A preferred anticoagulant is ethylenediaminetetraacetic acid (EDTA).

  Blood samples are evaluated using automated clinical analyzers (such as those manufactured by Coulter, Inc.) well known in the art, such as endpoints for measuring red blood cell synthesis and physiology (hematocrit (Hct), hemoglobin (Hgb) and total red blood cells. Number (RBC) etc.).

Synthesis of EPO-R agonist peptide homodimer of peptide monomer having amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1)
Step 1: Synthesis of Peptide Monomer Peptide monomers were prepared using standard Fmoc chemistry using TG-RAM resin (0.18 mmol / g, Rapp Polymere (Germany)) on an ABI 431A peptide synthesizer. To synthesize. For the synthesis of peptide monomers with an amidated carboxy terminus, the fully constructed peptide is cleaved from the resin using 82.5% TFA, 5% water, 6.25% anisole, 6.25% ethanedithiol. The deprotected product is filtered from the resin and precipitated with diethyl ether. After complete drying, the product is purified by C18 reverse phase high performance liquid chromatography using a gradient of acetonitrile / water in 0.1% trifluoroacetic acid. The structure of the peptide is confirmed by electrospray mass spectrometry. Peptide monomers can be shown as follows:

Step 2: Synthesis of trifunctional linker
To a solution of diethyliminoacetate (10.Og, 52.8 mmol) and Boc-beta-alanine (10.Og, 52.8 mmol) in 100 mL DCM was added diisopropylcarbodiimide (8.0 mL, 51.1 mmol) at room temperature over 10 minutes. Added in. During the addition, the reaction mixture was warmed to 10 degrees, cooled to room temperature over 20 minutes. The reaction mixture was stirred overnight and the precipitated diisopropylurea was filtered. The solvent was removed under reduced pressure to give a gum, the residue was dissolved in ethyl acetate and filtered again to remove further precipitated urea. The organic phase is placed in a separatory funnel, washed (saturated NaHCO ₃ , brine, 0.5N HCl, brine), dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give the diester product as a colorless oil. Got. The diester was mixed with a 1: 1 mixture of MeOH: THF (100 mL) to which water (25 mL) was added followed by NaOH (5 g, 125 mmol). The pH was measured to be> 10. The reaction mixture was stirred at room temperature for 2 hours and acidified to pH 1 with 6N HCl. The aqueous phase was saturated with NaCl and extracted four times with ethyl acetate. The combined organic phase was washed (brine), dried (MgSO ₄ ) and concentrated under reduced pressure to give a white semi-solid. The solid was dissolved in 50 mL DCM and 300 mL hexane was added to make a white slurry. The solvent was removed under reduced pressure to give the dibasic acid as a white solid (14.7 g, 91.5% yield over 2 steps). N-hydroxysuccinimide (770 mg, 6.69 mmol), diisopropylcarbodiimide (1.00 mL, 6.38 mmol) and 4-dimethylaminopyridine (3 mg, 0.02 mmol) against a solution of dibasic acid (1 g, 3.29 mmol) in 20 mL DMF ) Was added. The reaction mixture was stirred overnight and the solvent was removed under reduced pressure. The residue was mixed with ethyl acetate and filtered to remove the precipitated urea. The organic phase is placed in a separatory funnel, washed (saturated NaHCO ₃ , brine, 0.5N HCl, brine), dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give di- The NHS ester product (1.12 g, 68% yield) was obtained.

Step 3: Binding of trifunctional linker to peptide monomer For linker binding , 2 equivalents of peptide were mixed with 1 equivalent of trifunctional linker in dry DMF to obtain a clear solution, 2 minutes Later add 5 times equivalent of DIEA. The mixture is stirred at ambient temperature for 14 hours. The solvent is removed under reduced pressure and the crude product is dissolved in 80% TFA in DCM for 30 minutes to remove the Boc group and then purified by C18 reverse phase HPLC. The structure of the dimer is confirmed by electrospray mass spectrometry. By this coupling reaction, the linker is coupled to the nitrogen atom of the ε amino group of the lysine residue of each monomer. Binding using SEQ ID NO: 1 is shown below.

Step 4: Synthesis of a PEG moiety containing two linear PEG chains to which lysine is attached
mPEG2-Ricinol-NPC
Commercially available ricinol is treated with excess mPEG2-NPC to give MPEG2-ricinol, which is then reacted with NPC to form mPEG2-ricinol-NPC.
mPEG2-Lys-NHS
This product can be obtained commercially, for example, item number 2Z3X0T01 of Molecular Engineering Catalog (2003) of Nektar Therapeutics (35806 Huntsville Discovery Drive 490, Alabama).

Step 5: PEGylation of peptide dimer
The PEGylated peptide dimer through the carbamate linkage and the PEG species (mPEG ₂ -ricinol-NPC) are mixed in a 1: 2 molar ratio in dry DMF to give a clear solution. After 5 minutes, add 4 times equal amount of DIEA to this solution. The mixture is stirred at ambient temperature for 14 hours and then purified by C18 reverse phase HPLC. The structure of the PEGylated peptide is confirmed by MALDI mass spectrometry. The purified peptide was also applied for purification through cation exchange chromatography as outlined below. PEGylation using SEQ ID NO: 1 and mPEG-ricinol-NPC is shown below.

PEGylated peptide dimers through amide bonds and PEG species (mPEG ₂ -Lys-NHS from Shearwater, USA) are mixed in a 1: 2 molar ratio in dry DMF to give a clear solution. After 5 minutes, add 10-fold equivalent of DIEA to this solution. The mixture is stirred at ambient temperature for 2 hours and then purified by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass spectrometry. The purified peptide was also applied for purification through cation exchange chromatography as outlined below. PEGylation using SEQ ID NO: 1 and mPEG ₂ -Lys-NHS is shown below.

Step 6: Purification by Peptide Ion Exchange For some exchange supports, in addition to the ability to retain the starting dimeric peptide, separate the peptide-PEG bond from unreacted (or hydrolyzed) PEG. Investigate the ability to. Ion exchange resin (2-3 g) was loaded onto a 1 cm column, then converted to the sodium form (0.2 N NaOH was loaded onto the column until the pH of the eluent was 14; about 5 column volumes), then hydrogen Convert to mold (eluted with either 0.1 N HCl or 0.1 M HOAc until eluent matched packing pH; approximately 5 column volumes), then wash with 25% ACN / water until pH 6 did. Peptide or peptide-PEG conjugate before conjugation was dissolved in 25% ACN / water (10 mg / mL), pH adjusted to less than 3 with TFA, and then loaded onto the column. After washing with 2-3 column volumes of 25% ACN / water and collecting 5 mL fractions, the peptide was released from the column by eluting with 0.1 M NH ₄ OAc in 25% ACN / water and again 5 mL Fractions were collected. Analysis by HPLC revealed which fractions contained the desired peptide. Analysis using an evaporative light scattering detector (ELSD) indicates that the peptide is not bound as a contaminant when the peptide is retained on the column and eluted with NH ₄ OAc solution (typically between fractions 4-10) Of PEG was not observed. When the peptide was eluted with the first wash buffer (generally the first two fractions), no desired PEG binding and excess PEG separation was observed.

  The following columns were successful in retaining both peptide and peptide-PEG linkages and were successful in purifying peptide-PEG linkages from unbound peptides.

In the synthesis step 1 of the EPO-R agonist peptide homodimer of the peptide monomer having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2), the peptide monomer synthesized is:
(AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2)
Except that it is an EPO-R agonist of a peptide monomer having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2) as described in Example 1. Synthesize peptide homodimers.

  When PEG is attached to the spacer through a carbamate linkage, the final product of this synthesis using SEQ ID NO: 2 can be structurally shown as follows:

  When PEG is attached to the spacer through an amide bond, the final product of this synthesis using SEQ ID NO: 2 can be shown structurally as follows:

In Vitro Activity Assays This example describes various in vitro assays useful in assessing the activity and efficacy of the EPO-R agonist peptides of the present invention. The results of these assays demonstrate that the novel peptides of the present invention bind to EPO-R and activate EPO-R signaling. Furthermore, the results of these assays show a surprising increase in EPO-R binding affinity and biological activity in the novel peptide composition compared to previously described EPO mimetic peptides.

EPO-R agonist peptide monomers and dimers are prepared according to the methods provided in Example 1 or 2. A series of in vitro activity assays (including reporter assays, proliferation assays, competitive binding assays, and C / BFU-e assays) are used to assess the potency of these peptide dimers. Further details on these four assays are given below.
The results of these in vitro activity assays are summarized in Table 2.

1. Reporter Assay This assay is based on a reporter cell from the mouse pre-B cell line, Baf3 / EpoR / GCSFR fos / lux. This reporter cell line expresses a chimeric receptor that includes from the extracellular portion of the human EPO receptor to the intracellular portion of the human GCSF receptor. This cell line is further transfected with a fos promoter driven luciferase reporter gene construct. As a result of activation of this chimeric receptor by the addition of an erythropoietic agent, the luciferase reporter gene is expressed. Thus, when luciferin, a luciferase substrate, is added, light is generated. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

Baf3 / EpoR / GCSFR fos / lux cells are 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (supernatant from a culture of WEHI-3 cells; ATCC number TIB-68), And cultured in DMEM / F12 medium (Gibco) supplemented with penicillin / streptomycin. Approximately 18 hours prior to the assay, cells are starved by transferring them to DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day of the assay, cells were washed once with DMEM / F12 medium supplemented with 10% FBS (no WEHI-3 supernatant) and then supplemented with 10% FBS (no WEHI-3 supernatant) Cells in DMEM / F12 medium in the presence of known concentrations of test peptides or using EPO (R & D Systems (Minneapolis, MN)) as a positive control, cells (1 × 10 ⁶ cells / Incubate mL). In this assay, serial dilutions of test peptides are tested simultaneously. The assay plate is incubated for 4 hours at 37 ° C. in a 5% CO ₂ atmosphere, after which luciferin (Steady-Glo; Promega, Madison, Wis.) Is added to each well. After a 5 minute incubation, the light emission is measured using a Packard Topcount Luminometer (Packard Instrument, Downers Grove, Ill.). Light counts are plotted against test peptide concentration and analyzed using Graph Pad software. The concentration of test peptide that results in a half-maximal emission of light is recorded as the EC50.

2. Proliferation assay This assay is based on the mouse pre-B cell line, Baf3, which is transfected to express human EPO-R. Growth of the resulting cell line, BaF3 / Gal4 / Elk / EPOR, depends on the activation of EPO-R. Quantify the degree of cell proliferation using MTT. At this time, the signal in the MTT assay is proportional to the number of viable cells.

BaF3 / Gal4 / Elk / EPOR cells are cultured in DMEM / F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC number TIB-68) in a spinner flask To do. Cultured cells are starved overnight at a cell density of 1 × 10 ⁶ cells / ml in DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant in a spinner flask. Next, the starved cells were washed twice with Dulbecco PBS (Gibco) and supplemented with 10% FBS (no WEHI-3 supernatant) in 1 × 10 ⁶ DMEM / F12 medium Resuspend to a cell density of / ml. Next, 50 μL aliquots (˜50,000 cells) of cell suspension are plated in triplicate in 96 well assay plates. Serial dilutions of test EPO mimetic peptides in 50 μL aliquots in DMEM / F12 medium supplemented with 10% FBS (no WEHI-3 Supernatant I), or 50 μL EPO (R & D Systems, Minneapolis, MN) )) Or Aranesp ^™ (darbepoetin α; an EPO-R agonist commercially available from Amgen) is added to a 96-well assay plate (final well volume: 100 μL). For example, 12 different dilutions can be tested, with a final concentration of test peptide (or control EPO peptide) ranging from 81 OpM to 0.0045 pM. The plated cells are then incubated for 48 hours at 37 ° C. Next, 10 μL of MTT (Roche Diagnostics) is added to each culture plate well and incubated for 4 hours. The reaction is then stopped by adding 10% SDS + 0.01N HCl. The plate is then incubated overnight at 37 ° C. Next, the absorbance of each well at a wavelength of 595 nm is measured by spectrophotometry. A plot of absorbance readings against test peptide concentration is constructed and the EC50 is calculated using Graph Pad software. The test peptide concentration at which the absorbance is half the maximum is recorded as the EC50.

3. Competitive binding assay
Competitive binding using an assay that produces a light signal as a function of the proximity of two beads: a streptavidin donor bead with a biotinylated EPO-R binding peptide tracer and an acceptor bead to which EPO-R binds Perform the calculation. Light is produced by non-radiative energy transfer. When irradiated with light, singlet oxygen is released from the first bead, and contact with the released singlet oxygen causes light emission from the second bead. A set of these beads is commercially available (Packard). Bead proximity is created by the binding of EPO-R binding peptide tracer to EPO-R. For binding to EPO-R, a test peptide that competes with the EPO-R binding peptide tracer will prevent this binding and cause a decrease in light emission.

More specifically, the method is as follows. 4 μL of serial dilutions of test EPO-R agonist peptide, or positive or negative controls, are added to wells of a 384 well plate. Then add 2 μL / well of receptor / bead cocktail. The receptor bead cocktail consists of: 15 μL of 5 mg / ml streptavidin donor beads (Packard); 15 μL of 5 mg / ml monoclonal antibody abl79 (this antibody is a human placental alkaline phosphatase protein contained in recombinant EPO-R Recognize part); acceptor beads coated with protein A (protein A binds to abl79 antibody; Packard); 112.5 μL of recombinant EPO-R diluted 1: 6.6 (containing abl79 target epitope) For a portion of human placental alkaline phosphatase protein, produced as a fusion protein in Chinese hamster ovary cells); and 607.5 μL Alphaquest buffer (4OmM HEPES, pH 7.4; 1 mM MgCl ₂ ; 0.1% BSA, 0.05% Tween 20). Tap to mix. Add 2 μL / well of biotinylated EPO-R binding peptide tracer (final concentration: 3OnM). Using SEQ ID NO: 4, the peptide tracer EPO-R binding peptide (see “Receptor EC50 (pM)” in the table) is prepared according to the method described in Example 1.

  Centrifuge for 1 minute to mix. Seal the plate with a Packard Top Seal and wrap in foil. Incubate overnight at room temperature. After 18 hours, the light emission is read using an AlphaQuest reader (Packard). Plot light emission against peptide concentration and analyze with Graph Pad or Excel.

  The concentration of test peptide that resulted in a 50% decrease in light emission compared to the absence of test peptide is recorded as the IC50.

4). C / BFU-e assay
EPO-R signaling stimulates bone marrow stem cells to differentiate and red blood cell progenitors to proliferate. This assay measures the ability of a test peptide to stimulate the proliferation and differentiation of erythroid progenitor cells from primary human bone marrow pluripotent stem cells.

For this assay, serial dilutions of test peptides are prepared in IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These serial dilutions, or positive control EPO peptide, are then added to methylcellulose to a final volume of 1.5 mL. The mixture of methylcellulose and peptide is then vortexed thoroughly. Thaw aliquots (100,000 cells / mL) of human bone marrow derived CD34 ⁺ cells (Poietics / Cambrex). In a 50 mL tube, gently add thawed cells to 0.1 mL lmg / ml DNAse (Stem Cells). Next, gently add 40-50 mL of IMDM medium to the cells. The medium is added drop-wise along the side of the 50 ml tube for the first 10 mL and the remaining medium is slowly dispensed along the side of the tube. The medium is then carefully removed by spinning the cells at 900 rpm for 20 minutes and gently aspirating. Resuspend cells in 1 ml IMDM medium and measure cell density per ml on hemacytometer slide (10 μL aliquot of cell suspension on slide; cell density is average number × 10,000 cells / mL) . Next, the cells are diluted with IMDM medium to a cell density of 15,000 cells / mL. Next, 100 μL of diluted cells is added to each 1.5 mL of methylcellulose and peptide sample (final cell concentration in assay medium is 1,000 cells / mL) and the mixture is vortexed. Remove bubbles in the mixture and aspirate 1 mL using a blunt needle. Add 0.25 mL of the aspirated mixture from each sample to each of the four wells of a 24-well plate (Falcon brand). The plated mixture is incubated for 14 days at 37 ° C. in 5% CO ₂ in a humid incubator. A phase contrast microscope (5-10x objective; final magnification 100x) is used to score for the presence of erythroid colonies. The test peptide concentration at which the number of colonies formed was 90% of the maximum compared to that observed with the EPO positive control is recorded as EC90 (see Table 2; C / BFU-e EC90).

In Vivo Activity Assays This example describes various in vivo assays useful in assessing the activity and efficacy of the EPO-R agonist peptides of the present invention. EPO-R agonist peptide monomers and dimers are prepared according to the method provided in Example 1. The in vivo activity of these peptide monomers and dimers is assessed using serial assays, including erythrocytosis / exhypoxic mouse bioassays and reticulocyte assays. Further details on these two assays are given below.

1. Bioassay of erythrocytosis / hypoxia mice (Exhypoxic)
The test peptides are assayed for in vivo activity in a bioassay of erythropoietic and exhypoxic mice to which the method described in Cotes and Bangham (1961), Nature 191: 1065-1067 has been applied. In this assay, the test peptide functions as an EPO mimetic, ie, tests the ability to activate EPO-R and induce new erythrocyte synthesis. Erythrocyte synthesis is quantified based on the incorporation of radiolabeled iron into the hemoglobin of synthesized erythrocytes.

  BDF1 mice are acclimated to ambient conditions for 7-10 days. Weigh all animals and do not use low-weight animals (<15 grams). Mice are applied to a continuous conditioning cycle in a low pressure chamber for a total of 14 days. Each 24-hour cycle consists of 18 hours of 0.40 ± 0.02% atmospheric pressure and 6 hours of ambient pressure. After conditioning, the mice are maintained at ambient pressure for an additional 72 hours prior to dosing.

  Test peptides or recombinant human EPO standards are diluted in PBS + 0.1% BSA vehicle (PBS / BSA). The peptide monomer stock solution is first solubilized in dimethyl sulfoxide (DMSO). The negative control group includes one group of mice injected with PBS / BSA alone and one group of mice injected with 1% DMSO. Each dosing group includes 10 mice. Mice are injected subcutaneously (neck) with 0.5 mL of the appropriate sample.

48 hours after sample injection, mice are injected intraperitoneally with 0.2 ml of Fe ⁵⁹ (Dupont, NEN) (dose: approximately 0.75 μ Curie per mouse). 24 hours after administration of Fe ^59, the mice were weighed, 48 hours after administration of the Fe ^59, mice are sacrificed. Blood is collected from each animal by cardiac puncture and the hematocrit value is measured (heparin was used as an anticoagulant). Each blood sample (0.2 ml) is analyzed for Fe ⁵⁹ uptake using a Packard gamma counter. Unresponsive mice (ie, mice with less radioactive uptake than the negative control group) are excluded from the appropriate data set. Mice with a hematocrit value less than 53% of the negative control group are also excluded.

  Results are obtained from a set of 10 animals for each experimental dose. Calculate the average amount of radioactivity (counts per minute (CPM)) incorporated into blood samples obtained from each group.

2. Reticulocyte Assay Normal BDF1 mice receive either EPO control or test peptide for 3 consecutive days (dose: 0.5 mL, subcutaneous injection). On day 3, iron dextran (100 mg / ml) is also administered to the mice (dose: 0.1 mL, intraperitoneal injection). On day 5, mice are anesthetized with CO ₂ and bled by cardiac puncture. The percentage of reticulocytes in each blood sample is determined by staining with thiazole orange and flow cytometry analysis (reticulocyte count program). Measure hematocrit manually. The corrected reticulocyte percentage is determined using the following formula:
% RETIC _{correction value (CORRECTED)} =% RETIC _{observation value (OBSERVED)} X (Hematocrit _{individual value (INDIVIDUAL)} / Hematocrit _{normal value (NORMAL)} )

3. Hematology Assay Normal CD1 mice are administered either an EPO positive control, a test peptide, or a vehicle by intravenous bolus injection for 4 weeks. Positive control and test peptide dose ranges (expressed in mg / kg) are tested by varying the active compound concentration in the formulation. The amount to be injected is 5 mg / kg. The vehicle control group consists of 12 animals, and each remaining dose group consists of 8 animals. Record daily viability and weekly body weight.

  The administered mice are fasted and anesthetized by isoflurane inhalation on day 1 (vehicle control mice) and on days 15 and 29 (4 animals / group / day), and terminal blood is obtained by cardiac puncture or abdominal aortic puncture. Collect the sample. Transfer blood to Vacutainer® brand tubes. A preferred anticoagulant is ethylenediaminetetraacetic acid (EDTA).

  Blood samples are evaluated using automated clinical analyzers (such as those manufactured by Coulter, Inc.) well known in the art, such as endpoints for measuring red blood cell synthesis and physiology (hematocrit (Hct), hemoglobin (Hgb) and total red blood cells. Number (RBC) etc.).

Peptide I is immunologically different from recombinant EPO and we have experimented to ameliorate anti-EPO antibody-induced anemia in a rat model of PRCA and have found that peptide I has slowed down with a long half-life It has been demonstrated to be a potent erythropoiesis stimulator with clearance time (Fan Q. et al. (2006) Experimental Hematology 34: 1303-1311). We have also demonstrated the potential of peptide I to improve PRCA associated with anti-EPO antibodies in humans (Woodburn et al. (2007) Experimental Hematology 35: 1201-1208).

  The potential for cross-reactivity is related to the possibility of treating existing cases of PRCA induced by anti-EPO antibodies with peptide I. We experimented with multiple species (including mice, rabbits, cynomolgus monkeys, and humans) to characterize the binding of anti-peptide I antibodies to recombinant EPO and the binding of anti-recombinant EPO antibodies to peptide I. It is carried out. Data from experiments conducted by the inventors showed that antibodies against recombinant EPO do not cross-react with peptide I, and conversely, antibodies against peptide I do not cross-react with recombinant EPO.

  The efficacy of ameliorating anemia caused by PRCA induced by recombinant EPO antibody of peptide I was evaluated in a rat model of PRCA. Experiments conducted by the inventors have shown that peptide I improved antibody-induced anemia in a rat model of PRCA. The rat model of PRCA consisted of inducing an anti-EPO antibody in the rat to produce a model of PRCA. Following induction of PRCA in rats, the erythropoietic activity of peptide I compared to rats treated with recombinant EPO was evaluated after weekly administration of peptide I or recombinant EPO in a rat model of PRCA. Over time, hemoglobin levels in the rat model of PRCA treated with recombinant EPO decreased from the rat model of PRCA treated with peptide I.

  Furthermore, the ability of peptide I to improve PRCA-induced anemia was evaluated by administering peptide I to rats positive for anti-EPO antibodies. Improvement of anemia was measured by a significant increase in reticulocyte increase in anti-EPO antibody positive anemia rats treated with peptide I compared to anti-EPO antibody positive anemia rats treated with recombinant EPO. In addition, anemia in anti-EPO positive animals treated with Peptide I recovered and resulted in higher hemoglobin levels than those obtained in anti-EPO antibody negative control rats.

  Experiments were also conducted to see if normal hemoglobin levels could be maintained in anemic anti-EPO antibody positive rats. Approximately every 4 weeks, anemic anti-EPO antibody positive rats were treated with a total of 7 additional injections with peptide I. Hemoglobin levels in antibody positive rats treated with Peptide I and vehicle were compared to simultaneous controls. The data collected demonstrated that repeated improvement of hemoglobin levels resulted from repeated administration of peptide I every 4 weeks to anemic anti-EPO antibody positive rats.

  The PRCA rat model is a suitable model because it showed similar results to those observed in patients with PRCA induced by anti-EPO antibodies. EPO-induced PRCA in humans and rats is associated with the presence of anti-EPO antibodies, the absence of reticulocytes, and a normal number of granulocyte and megakaryocyte lineages coexisting with a limited number of bone marrow erythroid progenitors. Characterized by associated severe anemia. Peptide I ameliorated EPO-induced anemia in these animals.

Treatment of PRCA mediated by anti-EPO antibody with peptide I
1.1 Study Methods An open-label, prospective, non-randomized study was conducted to examine the ability of peptide I to improve anemia in patients with PRCA characterized by anti-EPO antibodies. The primary objective previously identified was that hemoglobin exceeding 11 g / dL using peptide I in patients with PRCA mediated by anti-EPO antibodies without the need for erythrocyte transfusions during a 6-month treatment period (KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for Anemia in Chronic Kidney Disease (2006) Am. J. Kidney Dis. 47 (Suppl 3): S11-S145.] And European guidelines [Locatelli F et al. (2004) Nephrol. Dial. Transplant 19 (Suppl 2): iil-47.], Both of which are met). We also evaluated the effect of peptide I on reducing erythrocyte transfusion, the time needed to improve anemia without transfusion, the required dose, and safety.

  Investigators and sponsors design, conduct and oversee the study, consider and confirm that all patients are eligible to participate in the study, and obtain monthly safety and efficacy data. It was examined. The trial protocol was approved by the national health department and local ethics committees in the UK, France and Germany. All patients submitted written informed consent, and the trial was conducted in accordance with the Good Clinical Practice guidelines on the conduct of pharmaceutical clinical trials and the Declaration of Helsinki. The investigator obtained the data, the development contractor monitored the data, and the investigator and the executing company were responsible for data analysis.

1.2 Patient eligibility
Patients aged over 18 years with PRCA or erythropoiesis dysfunction due to CKD (whether dialysis is required) and anti-EPO antibodies were eligible for the trial. Patients needed to have the following documented medical history: (1) While receiving stable or gradually increasing doses of protein-based ESA or depending on erythrocyte transfusion (2) Reticulocyte count is less than 30 × 10 ⁹ / L; (3) Bone marrow examination showed severe hypoerythropoiesis or no erythropoiesis; and (4 ) Test confirmed positive anti-EPO antibody. Patients may have received immunosuppressive therapy in advance, but such medications should have been interrupted for at least 3 months before participating in the trial. At study participation, patients were either dependent on blood transfusions or required that hemoglobin levels continue to be below 11 g / dL without ESA therapy. The main exclusion criteria included having another hematological disorder, another known cause of PRCA, and current ESA or immunosuppressive therapy.

1.3 Treatment and measurement of results Blood samples were taken for baseline measurements of total blood count, reticulocyte count, urea, electrolytes, liver function tests, iron status, anti-EPO antibody, and anti-peptide I antibody. Consistent with study eligibility criteria, treatment with peptide I started at a dose of 0.05 mg / kg and was injected subcutaneously every 4 weeks. While hemoglobin and reticulocyte counts were performed weekly, samples were taken monthly for other safety testing parameters (including urea, electrolytes, liver function tests, anti-EPO and anti-peptide I antibodies). The frequency and dose of each injection was adjusted based on the patient's hemoglobin response to achieve a target range of 11-13 g / dL for hemoglobin levels. Subsequently, this target range is determined by CREATE [Drueke TB et al. (2006) N. Engl. J. Med. 355: 2071-84] and CHOIR [Singh AK et al. (2006) N. Engl. J. Med 355: 2085. -98] following the publication of the KDOQI recommendation [KDOQI Clinical Practice Guideline and Clinical Practice Recommendations for Anemia in Chronic Kidney Disease (2007) Am. J. Kidney Disease 50: 471-530] It was changed to 11-12g / dL at the beginning of 2007.

  The anti-peptide I antibody assay uses an ELISA method with a sensitivity of 300 ng / mL (Fan Q. et al. (2006) Exp. Hematol. 34: 1303-11; and Woodburn KW et al. (2007) Exp. Hematol 35: 1201-8), conducted at the laboratory of the implementing company. Anti-EPO antibody was measured by radioimmunoprecipitation assay. To confirm that patient antibodies do not cross-react with peptide I, these patient sera were tested in bone marrow cultures (established from healthy human donors as previously described [Verhelst D et al., (2004) Lancet 363: 1768-71]). Culture conditions included either 20% fetal calf serum (as a control) or one of the study patient sera. Cultures contained either 1 IU / mL recombinant human EPO or 0.55 μg / mL Peptide I (concentrations previously indicated correspond to 1 IU / mL EPO due to red blood cell increase Need it). On day 7, red blood cell colonies were counted.

1.4 Statistical analysis The investigator will estimate the maximum number of patients who may be eligible to access the study center, and based on that, 5-20 patients with PRCA mediated by anti-EPO antibodies should be included The trial was designed. Patients were treated during the first 6 months. If the patient responded satisfactorily, peptide I administration was continued for a maximum of 18 months. All patients who received at least one dose of Peptide I were included in the safety analysis, and patients whose hemoglobin levels were measured at least once following administration were included in the efficacy analysis. All safety and efficacy results were compiled using descriptive statistics. Data are expressed as median, first quartile (25th percentile), third quartile (75th percentile) and range.

2.1 Patient results and baseline characteristics
Ten patients were eligible for the trial and enrolled one after another between March 2006 and February 2007. There were five in Germany, three in the UK, and two in France. Results reflect the data collected and reported on average 13.5 months of follow-up (range: 3-17 months) until August 23, 2007. Nine of the patients received dialysis (7 had hemodialysis and 2 had peritoneal dialysis), and 1 did not require renal replacement therapy. The ratio of patients to men and women was 8: 2. The median age at the start of the trial was 65 years (range: 28-92 years). At study entry, nine patients were dependent on blood transfusions, and the same nine patients had laboratory values that showed severe iron overload due to repeated transfusions and poor iron utilization. Hematological parameters at baseline were as follows: median hemoglobin 9.7 g / dL (range: 8.3 to 12.6 g / dL); median reticulocyte count 21.3 × 10 ⁹ / L (range: 1.8) ~70 × 10 ⁹ / L); median ferritin 1,356μg / L (range: 229~7,496μg / L); and median 70.9% transferrin saturation (TSAT) (range: 23-91%). All patients had confirmed anti-EPO antibodies. Three patients (# 3, # 4 and # 6 in Table 1) were originally described for antibody-mediated PRCA (Rossert J et al. (2004) J. Am. Soc. Nephrol. 15 : 398-406; Casadevall N et al. (2004) ASN, American Society of Nephrology no. SU-P0060), did not show complete suppression of erythropoiesis. Two of these patients were found early during the onset (# 3) or recurrence (# 4, # 6) of the syndrome and had signs of decreased erythropoiesis in the presence of anti-EPO antibodies. And was excluded from further treatment with conventional ESAs. The bone marrow in patient # 3 showed erythropoiesis dysplasia (rather than aplasia), and this patient had not yet received a blood transfusion. Patient # 4 had previously been diagnosed with PRCA and relapsed from re-exposure to epoetin, increasing anti-EPO antibody levels and decreasing hemoglobin levels. Patient # 6 had a definitive diagnosis of PRCA 6 months ago and relied on blood transfusions, and the reticulocyte count was 31-70 × 10 ⁹ / L prior to study entry. The demographic and baseline characteristics of the 10 patients enrolled in the trial are summarized in the following table:

* Patient # 6 received red blood cell transfusions monthly before the trial; the reticulocyte count was as low as 2.2 × 10 ⁹ / L for 4 months prior to study entry. The baseline values reported here were obtained from blood samples taken approximately one month after transfusion; the results were high compared to the patient's medical history.
† Patient # 7 received a red blood cell transfusion every month prior to study entry; baseline hemoglobin values reported here were obtained 8 days after the patient received the blood transfusion.
‡ Baseline reticulocyte count for patient # 7 reflects the value obtained one week after the first peptide I injection; no actual baseline value (before treatment) was obtained.

2.2 Results of bone marrow culture
In the presence of 1 IU / mL EPO, patient sera completely inhibited red blood cell increases for all three patients evaluated (FIG. 1). On the other hand, when peptide I (0.55 μg / mL) was added to the culture, erythroid differentiation was observed under the same conditions.

2.3 Treatment of anemia The hemoglobin response of all 10 patients was defined as achieving and maintaining a hemoglobin concentration of more than 11 g / dL without blood transfusion at an average follow-up of 13.5 months. The median hemoglobin increased from 9.7 g / dL at baseline (with support from erythrocyte transfusion) to 11.6 g / dL at 6 months (FIG. 2). The median time for hemoglobin response was 10 weeks (range: 7-24 weeks). All patients showed reticulocyte response after 2 weeks per injection of peptide I, peak reticulocyte count is compared with a median 21.3 × 10 ⁹ / L from the baseline value, 100 × 10 ⁹ / L~ The range was 250 × 10 ⁹ / L (see FIG. 3). After treatment with Peptide I, the need for blood transfusion decreased and eventually disappeared. During the first month, three patients received red blood cell transfusions; at month 4, one patient was still receiving blood transfusions; at month 6, no patients needed blood transfusions And at month 7, one patient received a blood transfusion immediately prior to kidney transplant surgery. No patients needed further blood transfusion after the 8th month (see FIG. 2). To achieve this response, all patients needed a dose increase from baseline, and one patient received Peptide I temporarily every two weeks. The median dose during the hemoglobin reaction was 0.08 mg / kg per month (range: 0.075 to 0.2 mg / kg). At the time of data cut-off, the median ferritin level decreased to 1,243 μg / L (range: 168 to 4,654 μg / L), and the median TSAT decreased to 50.0% (range: 31.1 to 90%).

  After up to 10 doses (median: 6 doses, range: 4-10 doses), no anti-peptide I antibody was detected in any of the 10 patient serum samples. Seven of the 10 patients were still anti-EPO antibody positive, but in most patients anti-EPO antibody decreased during the trial (data not shown).

  Peptide I injections are generally well tolerated, of the 108 events reported for 10 patients, 97 are mild or moderate in severity, and only 4 events are Reported: at least peptide I-may be related to hypertension (n = 2); severe bone pain not requiring treatment (n = 1); and hematoma at injection site (n = 1) ). Four severe adverse events were reported in two patients (one patient had endocarditis and arteriovenous fistula surgery; the other patient had atrial flutter and femoral aneurysm But none of these events were considered related to study drug. For the other three patients, severe, unrelated adverse events were reported (surgery for fatigue, headache, tinnitus, and Dupuytren contracture). During the study period, 3 patients received kidney transplants and were dropped out of the study for this reason and were censored at the 3rd, 6th, and 6th months, respectively.

3.1 Conclusions This trial provides data indicating that in the presence of circulating neutralizing anti-EPO antibodies, peptide I can rescue patients with antibody-mediated PRCA as a result of their ability to stimulate erythropoiesis. provide. All 10 patients achieved the primary endpoint of increasing hemoglobin to a level higher than 11 g / dL without blood transfusion. Peptide I was well tolerated by subjects in the trial and no serious adverse events related to peptide I were reported.

  Patients enrolled in this trial created functionally related anti-EPO antibodies during treatment with either epoetin alfa, epoetin beta, or darbepoetin alfa. Four patients had transfusion-dependent anti-EPO antibody-mediated PRCA for several years prior to enrollment. Two patients had a relapse of PRCA due to re-exposure to epoetin following a decrease in antibody titer. Other patients were diagnosed with the disease shortly before entering the trial. In anticipation of enrollment in trials, these patients were not treated with immunosuppressive therapy. In all patients, due to the presence of anti-EPO antibodies, further treatment with protein-based ESA was not possible. Therefore, in some patients, early exposure to epoetin resulted in a decrease in anti-EPO levels and erythropoiesis in the bone marrow (rather than complete suppression of erythropoiesis). Prior to initiation, little or no red blood cell transfusion was received. However, in contrast to epoetin or darbepoetin alfa, the action of peptide I is not neutralized by anti-EPO antibodies. This is due to the in vitro data obtained in this trial (see Figure 3) and previous in vitro and animal experiments (Woodburn KW et al. (2007) Exp. Hematol. 35: 1201-8; and Casadevall N et al. (2004). ASN, American Society of Nephrology no. SU-P0060).

  Overall, peptide I is well tolerated and provides a new treatment option in patients with PRCA caused by neutralizing anti-EPO antibodies.

[Sequence Listing]
<210> 1
<211> 20
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> Xaa is N-acetylglycine
<220>
<221> MISC_FEATURE
<222> (13) .. (13)
<223> Xaa is 1-naphthylalanine
<400> 1
Xaa Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln
1 5 10 15

Pro Leu Arg Lys
20

<210> 2
<211> 21
<212> PRT
<213> artificial
<220>
<223> synthetic protein

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> Xaa is N-acetylglycine
<220>
<221> MISC_FEATURE
<222> (13) .. (13)
<223> Xaa is 1-naphthylalanine
<220>
<221> MISC_FEATURE
<222> (20) .. (20)
<223> Xaa is N-methylglycine
<400> 2
Xaa Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln
1 5 10 15

Pro Leu Arg Xaa Lys
20

<210> 3
<211> 20
<212> PRT
<213> artificial
<220>
<223> synthetic protein

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> Xaa is N-acetylglycine
<220>
<221> MISC_FEATURE
<222> (13) .. (13)
<223> Xaa is 1-naphthylalanine
<220>
<221> MISC_FEATURE
<222> (20) .. (20)
<223> Xaa is N-methylglycine
<400> 3
Xaa Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln
1 5 10 15

Pro Leu Arg Xaa
20

<210> 4
<211> 20
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> biotinylated
<400> 4
Gly Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Trp Val Cys Gln
1 5 10 15

Pro Leu Arg Gly
20

<210> 5
<211> 5
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> Xaa is N-acetylglycine
<220>
<221> MISC_FEATURE
<222> (4) .. (4)
<223> Y side chain tBu protected
<400> 5
Xaa Gly Leu Tyr Ala
1 5

<210> 6
<211> 4
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> NH2 protected by Fmoc
<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> side chain Acm protected
<220>
<221> MISC_FEATURE
<222> (2) .. (2)
<223> side chain Trt protected
<400> 6
Cys His Met Gly
1

<210> 7
<211> 8
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> NH2 protected by Fmoc
<220>
<221> MISC_FEATURE
<222> (3) .. (3)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (4) .. (4)
<223> Xaa is 1-naphthylalanine
<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> side chain Acm protected
<220>
<221> MISC_FEATURE
<222> (7) .. (7)
<223> side chain Trt protected
<400> 7
Pro Ile Thr Xaa Val Cys Gln Pro
1 5

<210> 8
<211> 3
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (2) .. (2)
<223> side chain Pbf protected
<220>
<221> MISC_FEATURE
<222> (3) .. (3)
<223> Xaa is N-methylglycine
<220>
<221> MISC_FEATURE
<222> (3) .. (3)
<223> COOH Bn protected
<400> 8
Leu Arg Xaa
1

<210> 9
<211> 9
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> Xaa is N-acetylglycine
<220>
<221> MISC_FEATURE
<222> (4) .. (4)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> side chain Acm protected
<220>
<221> MISC_FEATURE
<222> (7) .. (7)
<223> side chain Trt protected
<400> 9
Xaa Gly Leu Tyr Ala Cys His Met Gly
1 5

<210> 10
<211> 11
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> NH2 protected by Fmoc
<220>
<221> MISC_FEATURE
<222> (3) .. (3)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (4) .. (4)
<223> Xaa is 1-naphthylalanine
<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> side chain Acm protected
<220>
<221> MISC_FEATURE
<222> (7) .. (7)
<223> side chain Trt protected
<220>
<221> MISC_FEATURE
<222> (10) .. (10)
<223> side chain Pbf protected
<220>
<221> MISC_FEATURE
<222> (11) .. (11)
<223> Xaa is N-methylglycine
<220>
<221> MISC_FEATURE
<222> (11) .. (11)
<223> COOH Bn protected
<400> 10
Pro Ile Thr Xaa Val Cys Gln Pro Leu Arg Xaa
1 5 10

<210> 11
<211> 20
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> Xaa is N-acetylglycine
<220>
<221> MISC_FEATURE
<222> (4) .. (4)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> side chain Acm protected
<220>
<221> MISC_FEATURE
<222> (7) .. (7)
<223> side chain Trt protected
<220>
<221> MISC_FEATURE
<222> (12) .. (12)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (13) .. (13)
<223> Xaa is 1-naphthylalanine
<220>
<221> MISC_FEATURE
<222> (15) .. (15)
<223> side chain Acm protected
<220>
<221> MISC_FEATURE
<222> (16) .. (16)
<223> side chain Trt protected
<220>
<221> MISC_FEATURE
<222> (19) .. (19)
<223> side chain Pbf protected
<220>
<221> MISC_FEATURE
<222> (20) .. (20)
<223> Xaa is N-methylglycine
<220>
<221> MISC_FEATURE
<222> (20) .. (20)
<223> COOH Bn protected
<400> 11
Xaa Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln
1 5 10 15

Pro Leu Arg Xaa
20

<210> 12
<211> 20
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> Xaa is N-acetylglycine
<220>
<221> MISC_FEATURE
<222> (4) .. (4)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> side chain Acm protected
<220>
<221> MISC_FEATURE
<222> (7) .. (7)
<223> side chain Trt protected
<220>
<221> MISC_FEATURE
<222> (12) .. (12)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (13) .. (13)
<223> Xaa is 1-naphthylalanine
<220>
<221> MISC_FEATURE
<222> (15) .. (15)
<223> side chain Acm protected
<220>
<221> MISC_FEATURE
<222> (16) .. (16)
<223> side chain Trt protected
<220>
<221> MISC_FEATURE
<222> (19) .. (19)
<223> side chain Pbf protected
<220>
<221> MISC_FEATURE
<222> (20) .. (20)
<223> Xaa is N-methylglycine
<400> 12
Xaa Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln
1 5 10 15

Pro Leu Arg Xaa
20

<210> 13
<211> 20
<212> PRT
<213> artificial
<220>
<223> synthetic peptide

<220>
<221> MISC_FEATURE
<222> (1) .. (1)
<223> Xaa is N-acetylglycine
<220>
<221> MISC_FEATURE
<222> (4) .. (4)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (7) .. (7)
<223> side chain Trt protected
<220>
<221> MISC_FEATURE
<222> (12) .. (12)
<223> side chain tBu protected
<220>
<221> MISC_FEATURE
<222> (13) .. (13)
<223> Xaa is 1-naphthylalanine
<220>
<221> MISC_FEATURE
<222> (16) .. (16)
<223> side chain Trt protected
<220>
<221> MISC_FEATURE
<222> (19) .. (19)
<223> side chain Pbf protected
<220>
<221> MISC_FEATURE
<222> (20) .. (20)
<223> Xaa is N-methylglycine
<400> 13
Xaa Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln
1 5 10 15

Pro Leu Arg Xaa
20

  The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing specification and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

In addition, it should be understood that all values are approximate and are provided for description.
Numerous references, including patents, patent applications, and various publications, are cited and discussed in the description of the present invention. The citation and / or discussion of such references is provided merely for clarity of explanation of the invention and any such reference is deemed to be “prior art” of the invention. is not. All references cited and discussed herein are hereby incorporated by reference in their entirety and are equally effective as if individually incorporated by reference to each reference. Have.

FIG. 1 shows a comparison of data from three patients with that of a control subject for inhibition of erythropoiesis using patient serum in the presence of EPO or peptide I. The light gray bars represent the number of red blood cell colonies in vitro when using control or patient sera in the presence of EPO. The dark gray bar represents the number of red blood cell colonies in vitro when using control or patient serum in the presence of peptide I. FIG. 2 shows the average hemoglobin (Hgb) concentration and the percentage of patients receiving erythrocyte transfusion after treatment with Peptide I. Gray bars indicate the percentage of patients receiving blood transfusions every month prior to treatment with peptide I (-3 to 0 months) and up to 9 months of treatment. FIG. 3 shows the change in the median reticulocyte count after treatment with peptide I. Arrows represent peptide I injections every month. The black line represents the median absolute number of reticulocytes, and the error bar represents the intra-quartile range. FIG. 4 shows the change in mean Hgb after receiving red blood cell transfusion without treatment with Peptide I in one patient. Arrows represent blood transfusions. FIG. 5 shows the change in mean Hgb and median reticulocyte count in one patient after erythrocyte transfusion and treatment with peptide I. The down arrow represents blood transfusion and the up arrow represents treatment with peptide I.

Claims (18)

A method of treating a patient having a disorder characterized by an anti-erythropoietin (EPO) antibody comprising administering to the patient a therapeutically effective amount of a compound according to Formula I that binds to and activates an erythropoietin receptor (EPO-R) . 2. The method of claim 1 wherein the therapeutically effective amount is a dose of 0.05 to 0.3 mg of the compound per kg patient body weight. The method according to claim 1, wherein the disorder is erythroblastic fistula (PRCA). The method according to claim 1 or 2, wherein the disorder is erythropoiesis. 2. The method of claim 1, wherein the patient has not previously been administered EPO or recombinant EPO. 3. The method of claim 1 or 2, wherein the patient has previously been administered EPO or recombinant EPO. A method of preventing a disorder characterized by an anti-EPO antibody comprising administering to a patient a therapeutically effective amount of a compound according to Formula I that binds to and activates an erythropoietin receptor (EPO-R). 8. The method of claim 7, wherein the therapeutically effective amount is a dose of 0.05 to 0.3 mg of the compound per kg patient body weight. The method according to claim 7 or 8, wherein the disorder is erythroblastosis. 9. The method of claim 7 or 8, wherein the patient has not previously been administered EPO or recombinant EPO. 9. The method of claim 7 or 8, wherein the patient has previously been administered EPO or recombinant EPO. A method for ameliorating anemia in a patient having a disorder characterized by an anti-EPO antibody comprising administering to the patient a therapeutically effective amount of a compound according to Formula I that binds to and activates the erythropoietin receptor (EPO-R). 13. The method of claim 12, wherein the therapeutically effective amount is a dose of 0.05 to 0.3 mg of the compound per kg patient body weight. 14. The method of claim 12 or 13, wherein ameliorating anemia comprises restoring hemoglobin to 10-13 g / dL in the patient. To improve the anemia in a patient, the hemoglobin was restored to 10~13g / dL, the number of reticulocytes to ^{100 × 10 9 / L~250 × 10} 9 / L comprising restoring, in claim 12 or 13 The method described. 14. The method of claim 12 or 13, wherein the anemia is anemia induced by chemotherapy. 14. The method of claim 12 or 13, wherein the anemia is anemia in a patient undergoing dialysis or pre-dialysis chronic kidney disease (CDK). 13. A method according to any of claims 1, 7 or 12, wherein the dose is administered once every 4 weeks.